Ethanol production in microorganisms

ABSTRACT

The present disclosure relates to methods and compositions for engineering photoautotrophic organisms to convert carbon dioxide and light into fatty acid esters and other molecules, including biofuels. The molecules are then secreted by the organism into a growth medium.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/403,938, filed Feb. 23, 2012, which is a continuation of U.S.application Ser. No. 13/243,194, now U.S. Pat. No. 8,216,816, filed Sep.23, 2011, which is a continuation of U.S. application Ser. No.13/166,601, now U.S. Pat. No. 8,048,666, filed Jun. 22, 2011, which is adivisional application of U.S. application Ser. No. 13/054,470, filedJan. 21, 2011, now U.S. Pat. No. 7,968,321, which is a national phaseentry of PCT/US2009/055949, filed on Sep. 3, 2009, which claims thebenefit of U.S. Provisional Application No. 61/184,757, filed Jun. 5,2009, U.S. Provisional Application No. 61/121,532, filed Dec. 12, 2008,U.S. Provisional Application No. 61/106,543, filed Oct. 17, 2008, andwhich is also a continuation-in-part of PCT/US2009/035937, filed Mar. 3,2009. The content of each these applications is incorporated herein byreference, for all purposes.

SEQUENCE LISTING

This application includes a Sequence Listing submitted electronically asa text file named “20593_US_Sequence_Listing.txt,” created on Feb. 23,2012, with a size of 92 kilobytes. The sequence listing consists of 29sequences and is incorporated by reference.

FIELD

The present disclosure relates to methods and compositions forengineering photoautotrophic organisms to convert carbon dioxide andlight into fatty acid esters and other molecules which are then secretedby the organism into a growth medium.

BACKGROUND

Photosynthesis is a process by which biological entities utilizesunlight and CO₂ to produce sugars for energy. Photosynthesis, asnaturally evolved, is an extremely complex system with numerous andpoorly understood feedback loops, control mechanisms, and processinefficiencies. This complicated system presents likely insurmountableobstacles to either one-factor-at-a-time or global optimizationapproaches [Nedbal et al., Photosynth Res., 93(1-3):223-34 (2007);Salvucci et al., Physiol Plant., 120(2):179-186 (2004); Greene et al.,Biochem J., 404(3):517-24 (2007)].

Existing photoautotrophic organisms (i.e., plants, algae, andphotosynthetic bacteria) are poorly suited for industrial bioprocessingand have therefore not demonstrated commercial viability for thispurpose. Such organisms have slow doubling time (3-72 hrs) compared toindustrialized heterotrophic organisms such as Escherichia coli (20minutes), reflective of low total productivities. In addition,techniques for genetic manipulation (knockout, over-expression oftransgenes via integration or episomic plasmid propagation) areinefficient, time-consuming, laborious, or non-existent.

SUMMARY

The invention described herein identifies pathways and mechanisms toconfer direct carbon-based products producing capacity tophotoautotrophic organisms. The resultant engineered carbon-basedproducts-producing photoautotrophs uniquely enable the efficientproduction of carbon-based products directly from carbon dioxide andlight, eliminating the time-consuming and expensive processing stepscurrently required to generate biofuels and biochemicals from biomasssources including corn, sugar cane, miscanthus, cellulose, and others.Accordingly, the novel microorganisms of the invention are capable ofsynthesizing carbon-based products of interest derived from variousbiosynthetic pathways by fixing CO₂ and are also capable of releasingsuch products.

Such products range from alcohols such as ethanol, propanol,isopropanol, butanol, fatty alcohols, fatty acid esters, wax esters;hydrocarbons and alkanes such as propane, octane, diesel, JP8; polymerssuch as terephthalate, 1,3-propanediol, 1,4-butanediol, polyols, PHA,PHB, acrylate, adipic acid, ε-caprolactone, isoprene, caprolactam,rubber; commodity chemicals such as lactate, DHA, 3-hydroxypropionate,γ-valerolactone, lysine, serine, aspartate, aspartic acid, sorbitol,ascorbate, ascorbic acid, isopentenol, lanosterol, omega-3 DHA,lycopene, itaconate, 1,3-butadiene, ethylene, propylene, succinate,citrate, citric acid, glutamate, malate, HPA, lactic acid, THF, gammabutyrolactone, pyrrolidones, hydroxybutyrate, glutamic acid, levulinicacid, acrylic acid, malonic acid; specialty chemicals such ascarotenoids, isoprenoids, itaconic acid; pharmaceuticals andpharmaceutical intermediates such as 7-ADCA/cephalosporin, erythromycin,polyketides, statins, paclitaxel, docetaxel, terpenes, peptides,steroids, omega fatty acids and other such suitable products ofinterest. Such products are useful in the context of fuels, biofuels,industrial and specialty chemicals, additives, as intermediates used tomake additional products, such as nutritional supplements,neutraceuticals, polymers, paraffin replacements, personal care productsand pharmaceuticals. These compounds can also be used as feedstock forsubsequent reactions for example transesterification, hydrogenation,catalytic cracking via either hydrogenation, pyrolisis, or both orepoxidations reactions to make other products.

A method of selecting and using various organisms to directly convertsunlight and carbon dioxide into carbon-based products is alsodisclosed. In one aspect of the invention, a method is provided tointroduce an engineered nucleic acid sequence encoding one or moreproteins capable of CO₂ fixation to produce and, in some examples,excrete or secrete carbon-based products of interest, such as ethanol,ethylene, hydrocarbons, ethyl esters and methyl esters. A salientfeature provided herein is that the microorganisms can produce variouscarbon-based products of interest preferably in commercial scale withoutthe need for a renewable carbon-based intermediate or source, such asbiomass, as a starting material.

To produce at least one of the desired carbon-based products of interestsuch as hydrocarbons, various organisms capable of CO₂ fixation, such asthose capable of photosynthesis or in the alternative, organismsengineered to fix CO₂ are used. For example, photoautotrophic organismsinclude eukaryotic plants and algae, as well as prokaryoticcyanobacteria, green-sulfur bacteria, green non-sulfur bacteria, purplesulfur bacteria, and purple non-sulfur bacteria.

In one aspect, a host cell capable of CO₂ fixation is engineered toproduce a carbon-based product having a desired number of carbons.Preferably the host cell produces various carbon-based products ofinterests in commercial scale.

In other examples a modified host cell is one that is geneticallymodified with an exongenous nucleic acid sequence encoding a singleprotein involved in a biosynthetic pathway involved in product orintermediate production. In other embodiments, a modified host cell isone that is genetically modified with exongenous nucleic acid sequencesencoding two or more proteins involved in a biosynthetic pathwayinvolved in product or intermediate production, for example, the firstand second enzymes in a biosynthetic pathway.

In another aspect, the present invention provides a host cell capable ofCO₂ fixation that produces ethylene. The host can include an exogenousnucleic acid encoding an ethylene forming enzyme, efe. The ethyleneforming enzyme, efe, can include a Ralstonia ethylene forming enzyme.

In one embodiment, such a carbon-based product of interest is ethanol.In a preferred embodiment, the host cell produces commercial yields ofethanol. Also provided is a method of using the organisms to directlyconvert sunlight, water and carbon dioxide directly into ethanol incommercial scale. A method of monetizing the ethanol produced as well asthe carbon dioxide taken up in association with the production ofethanol is additionally disclosed.

In some aspects, ethanol production is optimized by channeling carbonaway from glycogen and toward pyruvate, etc. during light exposure.Normally glycogen is formed in the light and it is consumed for reducingpower in the dark. In one embodiment, glycogen-synthesis genes areattenuated or knocked out and in other embodiments, glycolytic genes aremade constitutive. In other aspects, certain fermentative pathways, suchas those leading to acetate, lactate, succinate, etc., if present, areeliminated.

Still in other aspects, if light-dark cycle is to be implemented,glycogen production is optimize during light exposure (as opposed tobiomass, etc.) and increased in % of dry cell weight that can beglycogen (i.e., the cell is engineered to defeat any limitation thatkeeps the cells from swelling full of glycogen). Then, during the dark,ethanol synthesis is allowed to proceed from the accumulated glycogen,having attenuated or knocked out the other fermentative pathways.Furthermore, using a light-dark cycle that matches rates of glycogensynthesis/catabolism such that minimal time is wasted. is disclosed(glycogen doesn't run out and the cells sit unproductively in the dark,or there is too much glycogen to consume completely during the darkperiod).

In various aspects of the invention, described is a genetically modifiedphotosynthetic organism for sugar production. In certain embodiments,sugars, e.g., glucose, fructose or a combination thereof, are produced.Preferably, the sugars produced are diffused through uniporters ortransporters. In other embodiments, sugars are produced by expressingenzymes in a selected host cell producing 3-phosphoglyceraldehyde(3PGAL) and actively transported using transporters. In yet otherembodiments, photosynthetic organisms functionally lack cellulose,glycogen, or sucrose synthesis. The resulting photosynthetic products,e.g., sugars produced from the photosynthetic organisms, can be used asfeedstock or as a carbon source to produce additional carbon-basedproducts of interest.

In another aspect of the invention, the invention provides engineeredphotosynthetic organisms for producing maltose. In certain embodiments,the invention provides cloned genes for glycogen hydrolyzing enzymeswhich allow the engineered cells to hydrolyze glycogen to glucose and/ormaltose and transport maltose and glucose from the cell. Enzymes fortransporting maltose from the cell include the maltose efflux pump fromchloroplast for maltose transport: MEX1; glucose permeases, low and highKm, glucose:H+ symporter, glucose/fructose permease, general sugar:H+antiporter for glucose transport; and glucose 6-phosphate:Pi antiporter,triose-phosphate:phosphate antiporter for glucose-6-phosphate transportare contemplated transport mechanisms of the present invention.

In another embodiment, hydrocarbons are produced by engineering variousorganisms capable of CO₂ fixation or engineered to fix CO₂. In oneembodiment, the microorganisms are introduced with one or more exogenousnucleic acid sequences encoding acetyl-CoA:ACP transacylase activity(fabH), acetyl-CoA carboxylase activity (accBCAD), malonyl-CoA:ACPtransacylase activity (fabD), 3-ketoacyl-ACP synthase activity (fabB),3-ketoacyl-ACP reductase activity (fabG), 3-hydroxyacyl-ACP dehydrataseactivity (fabA), enoyl-ACP reductase activity (fabI), acyl-ACP hydrolaseactivity (FAS1), aldehyde dehydrogenase activity (adhA, adhB), alcoholdehydrogenase activity (ADH I), alkane 1-monooxygenase activity (alkB).

Additional genes that can be over-expressed for the production of fattyacid derivatives are for example, pdh, panK, aceEF (encoding the EIpdehydrogenase component and the E2p dihydrolipoamide acyltransferasecomponent of the pyruvate and 2-oxoglutarate dehydrogenase complexes,Accessions: NP_(—)414656, NP_(—)414657, EC: 1.2.4.1. 2.3.1.61,2.3.1.12), accABCD/fabH/fabD/fabG/acpP/fabF (encoding FAS, Accessions:CAD85557, CAD85558, NP 842277, NP 841683, NP 415613, EC: 2.3.1.180,2.3.1.39, 1.1.1.100, 1.6.5.3, 2.3.1.179), genes encoding fatty-acyl-coAreductases (Accessions: AAC45217, EC 1.2.1.-), UdhA or similar genes(encoding pyridine nucleotide transhydrogenase, Accession: CAA46822, EC:1.6.1.1) and genes encoding fatty-acyl-coA reductases (Accessions:AAC45217, EC 1.2.1.-).

In contrast to expressing exogenous nucleic acid sequences that allowfor the production of fatty acid derivatives, the host can have one ormore endogenous genes functionally deleted or attenuated. For example,ackA (EC 2.7.2.1), ackB (EC 2.7.2.1), adhE (EC 1.1.1.1, 1.2.1.10), fabF(EC 2.3.1.179), fabR (accession NP_(—)418398), fadE (EC 1.3.99.3,1.3.99.-), GST (EC 6.3.2.3), gpsA (EC 1.1.1.94), IdhA (EC 1.1.1.94),pflB (EC 2.3.1.54), plsB (EC 2.3.1.15), poxB (EC 1.2.2.2), pta (EC2.3.1.8), glutathione synthase (EC 6.3.2.3) and combinations thereof canbe at least attenuated.

Such microorganisms can be engineered to produce hydrocarbons or fattyacid of defined carbon chain length, branching, and saturation levels.

In some embodiments, peptides, e.g., thioesterase encoded by theexogenous nucleic acid sequences is expressed to provide homogeneousproducts, which would decreases the overall cost associated withfermentation and separation.

In some embodiments, the microorganisms include one or more exogenousengineered nucleic acids encoding acyl-CoA synthetase (EC6.2.1.3),thioesterase (EC 3.1.2.14), wax synthase (EC 2.3.1.75), alcoholacetyltransferase (EC 2.3.1.84) or a combination thereof. In otherembodiments, the microorganisms include engineered nucleic acidsencoding thioesterase (EC 3.1.2.14), acyl-CoA reductase (EC 1.2.1.50),alcohol dehydrogenase (EC 1.1.1.1), fatty alcohol forming acyl-CoAreductase (EC 1.1.1.*) or a combination thereof.

In some embodiments, the microorganisms described herein produce atleast 1 mg of carbon-based product of interest, e.g., hydrocarbon perliter of fermentation media. In more preferred embodiments, themicroorganisms produce at least 100 mg/L, 500 mg/L, 1 g/L, 5 g/L, 10g/L, 20 g/L, 25 g/L, 30 g/L, 35 g/L, 40 g/L, 50 g/L, 100 g/L, or 120 g/Lof hydrocarbons. In some examples, the hydrocarbon is produced andreleased from the microorganism and in yet other examples themicroorganism is lysed prior to separation of the product.

In some examples, the hydrocarbon includes a carbon chain that is atleast 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34carbons long. In some examples at least 50%, 60%, 70%, 80%, 85%, 90%, or95% of the hydrocarbon product made contains a carbon chain that is 2,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34 carbonslong. In yet other examples, at least 60%, 70%, 80%, 85%, 90%, or 95% ofthe fatty acid derivative product contain 1, 2, 3, 4, or 5, points ofunsaturation.

Certain biosynthetic pathways can be engineered to make fatty alcoholsand wax/fatty acid esters as illustrated in WO 2007/136762 (incorporatedby reference in its entirety for all purposes) the conversion of eachsubstrate (acetyl-CoA, malonyl-CoA, acyl-ACP, fatty acid, and acyl-CoA)to each product (acetyl-CoA, malonyl-CoA, acyl-ACP, fatty acid, andacyl-CoA), which can be accomplished using several differentpolypeptides that are members of the enzyme classes indicated.

Alcohols (short chain, long chain, branched or unsaturated) can beproduced by the hosts described herein. Such alcohols can be used asfuels directly or they can be used to create an ester, i.e. the A sideof an ester as described above. Such ester alone or in combination withthe other fatty acid derivatives described herein are useful a fuels.

Similarly, hydrocarbons produced from the microorganisms describedherein can be used as biofuels. Such hydrocarbon based fuels can bedesigned to contain branch points, defined degrees of saturation, andspecific carbon lengths. When used as biofuels alone or in combinationwith other fatty acid derivatives the hydrocarbons can be additionallycombined with additives or other traditional fuels (alcohols, dieselderived from triglycerides, and petroleum based fuels).

In one embodiment, the invention provides an engineered microbial hostcell, wherein said engineered host cell comprises one or more engineerednucleic acids, and wherein said engineered host cell is capable of usinga minimum amount of light energy to synthesize a carbon-based product ofinterest directly from carbon dioxide and water, and wherein saidcarbon-based product of interest is selected from the group consistingof: ethyl ester, methyl ester, sucrose, alcohol, ethanol, propanol,isopropanol, butanol, fatty alcohols, fatty acid ester, wax ester,hydrocarbons, n-alkanes, propane, octane, diesel, JP8, polymers,terephthalate, polyol, 1,3-propanediol, 1,4-butanediol, PHA, PHB,acrylate, adipic acid, ε-caprolactone, isoprene, caprolactam, rubber,lactate, DHA, 3-hydroxypropionate, γ-valerolactone, lysine, serine,aspartate, aspartic acid, sorbitol, ascorbate, ascorbic acid,isopentenol, lanosterol, omega-3 DHA, lycopene, itaconate,1,3-butadiene, ethylene, propylene, succinate, citrate, citric acid,glutamate, malate, HPA, lactic acid, THF, gamma butyrolactone,pyrrolidones, hydroxybutyrate, glutamic acid, levulinic acid, acrylicacid, malonic acid, carotenoid, isoprenoid, itaconic acid, limonene,pharmaceutical or pharmaceutical intermediates, erythromycin7-ADCA/cephalosporin, polyketides, statin, paclitaxel, docetaxel,terpene, peptide, steroid, and an omega fatty acid.

In another embodiment, the engineered nucleic acid comprised by the hostcell encodes an ethylene forming enzyme (Efe) activity. In a relatedembodiment, the ethylene forming enzyme is selected from Pseudomonassyringae pv. Phaseolicola D13182, P. syringae pv. Pisi AF101061 andRalstonia solanacearum AL646053. In another embodiment, the engineerednucleic acid encodes a codon-optimized Ralstonia ethylene-formingenzyme. In a related embodiment, the codon optimization is forEschericia coli codon usage. In yet another related embodiment, theengineered nucleic acid is SEQ ID NO. 7.

In certain embodiments, the engineered cell of the invention producesethylene in an amount greater than about 1 mg, 100 mg, 500 mg, 1 g, 5 g,10 g, 20 g, 25 g, 30 g, 35 g, 40 g, 50 g, 100 g, 120 g, or 150 g perliter of fermentation medium

In certain embodiments, the engineered nucleic acid comprised by theengineered cell of the invention encodes an alcohol dehydrogenaseactivity. In related embodiments, the alcohol dehydrogenase activity isselected from Z. mobilis adhII, Z. mobilis adhII TS42 and Z. mobilisadhB activity. In another related embodiment, the engineered nucleicacid encodes a NADPH-dependent alcohol dehydrogenase activity. In yetanother embodiment, wherein the NADPH-dependent alcohol dehydrogenaseactivity is Moorella sp. HUC22-1 adhA.

In another embodiment, the engineered nucleic acid comprised by theengineered cell of the invention encodes a pyruvate decarboxylaseactivity. In a related embodiment, the pyruvate decarboxylase activityis selected from Z. palmae and Z. mobilis pdc activity.

In certain embodiments, the engineered cell of the invention, inculture, is capable of producing ethanol in a yield of at least about249 mg/L culture medium in 72 hours. In certain other embodiments, theyield is at least about 296 mg/L of ethanol over 72 hours. In stillother embodiments, the ethanol yield is between about 2.5 to about 5 g/Lculture medium-hr. In other embodiments, the level of acetaldehyde insaid culture after 72 hours is less than about 14 mg/L. In otherembodiments, the cell in culture produces at least about 36 mg/L ofethanol per OD, or at least about 47 mg/L of ethanol per OD.

In another embodiment, the engineered nucleic acid comprised by theengineered cell of the invention encodes a methionine synthase activity.

In another embodiment, the engineered nucleic acid comprised by theengineered cell of the invention encodes a phosphate transporteractivity selected from the group consisting of an E. coli sugarphosphate transporter UhpT (NP_(—)418122.1), an A. thalianagluose-6-phosphate transporter GPT1 (AT5G54800.1), and an A. thalianagluose-6-phosphate transporter GPT2 (AT1G61800.1).

In another embodiment, the engineered nucleic acid comprised by theengineered cell of the invention encodes a phosphatase enzyme activityselected from the group consisting of a H. sapiens glucose-6-phosphataseG6PC (P35575), an E. coli glucose-1-phosphatase Agp (P19926), an E.cloacae glucose-1-phosphatase AgpE (Q6EV19), and an E. coli acidphosphatase YihX (P0A8Y3).

In another embodiment, the engineered nucleic acid comprised by theengineered cell of the invention encodes a glucose/hexose transporteractivity selected from the group consisting of a H. sapiens glucosetransporter GLUT-1, -3, or -7 (P11166, P11169, Q6PXP3), a S. cerevisiaehexose transporter HXT-1, -4, or -6 (P32465, P32467, P39003), and a Z.mobilis glucose uniporter Glf (P21906). In a related embodiment, theengineered cell of the invention further comprising an engineerednucleic acid encoding a Glucose/fructose:H+ symporter, a GlcP BacteriaGlcP of Synechocystis sp. (P15729), a major glucose (or 2-deoxyglucose)uptake transporter GlcP Q7BEC, a hexose (glucose and fructose)transporter, a PfHT1 of Plasmodium falciparum 097467, a Glut-1transporter, or a Glut-2 transporter.

In another aspect, the engineered cell provided by the invention isattenuated in an enzyme activity selected from the group consisting of:cellulose synthase; glycogen synthase; sucrose phosphate synthase;sucrose phosphorylase; alpha-1,4-glucan lyase; and 1,4-alpha-glucanbranching enzyme. In a related aspect, the engineered cell furthercomprises an engineered nucleic acid encoding a phosphatase or ahexokinase activity.

In yet another embodiment, the invention provides an engineered cellcomprising a glycogen hydrolysis activity selected from the groupconsisting of alpha, beta, gamma amylases; glucoamylase; isoamylase;pullulanase; amylomaltase; amylo-alpha-1,6-glucosidase; phosphorylasekinase; and phosphorylase.

In yet another embodiment, the invention provides an engineered cellcapable of producing a sugar or sugar phosphate selected from the groupconsisting of glucose, glucose-6-phosphate, fructose-6-phosphate,maltose, and maltose phosphate. In a related embodiment, the cell iscapable of producing a sugar or sugar phosphate above its endogenouslevels and transporting said sugar outside of the cell. In yet anotherrelated embodiment, the cell produces a sugar selected from the group ofsugars consisting of glucose, maltose, fructose, sucrose, xylose,pentose, rhamnose, and arabinose. In certain aspects, the engineeredcell of the invention, in culture, is capable of producing sugar orsugar phosphate in an amount greater than about 1 mg, 100 mg, 500 mg, 1g, 5 g, 10 g, 20 g, 25 g, 30 g, 35 g, 40 g, 50 g, 100 g, 120 g, or 150 gper liter of fermentation medium.

In yet another embodiment, the engineered cell provided by the inventioncomprises an engineered nucleic acid encoding an activity selected fromthe group consisting of an acetyl-CoA acetyltransferase, AtoB, aβ-hydroxybutyryl-CoA dehydrogenase, a crotonase, a CoA dehydrogenase, aCoA-acylating aldehyde dehydrogenase (ALDH), and an aldehyde-alcoholdehydrogenase, AdhE. In still another embodiment, the cell comprises anengineered nucleic acid encoding an activity selected from the groupconsisting of 2-dehydro-3-deoxyphosphoheptonate aldolase, aroF (EC2.5.1.54), a 3-dehydroquinate synthase, aroB (EC 4.2.3.4), a3-dehydroquinate dehydratase, aroD (EC 4.2.1.10), a 3-dehydroshikimatedehydratase, quiC (EC 4.2.1.n), a β-ketoadipyl-CoA synthase, pcaF (EC2.3.1.174), a β-ketoadipate CoA-transferase, pcalJ (EC 2.8.3.6), a3-oxoadipate enol-lactone hydrolase, pcaL (EC 3.1.1.24), a4-carboxymuconolactone decarboxylase, pcaL (EC 4.1.1.44), aγ-carboxy-cis,cis-muconate cycloisomerase, pcaB (EC 5.5.1.2), aprotocatechuate 3,4-dioxygenase, pcaGH (EC 1.13.11.3), a protocatechuate1,2-cis-dihydrodiol dehydrogenase, tpaC (EC 1.3.1.n), and aterephthalate 1,2-dioxygenase, tpaAB (EC 1.14.12.15). In yet anotherembodiment, the cell comprises an engineered nucleic acid encoding anactivity selected from the group consisting ofalpha-D-glucose-6-phosphate ketol-isomerase, PGI1 (EC 5.3.1.9), aD-Mannose-6-phosphate ketol-isomerase, din9 (EC 5.3.1.8), a D-Mannose6-phosphate 1,6-phosphomutase, atpmm (EC 5.4.2.8), a mannose-1-phosphateguanylyltransferase, cyt (EC 2.7.7.22), a GDP-mannose 3,5-epimerase, gme(EC 5.1.3.18), a galactose-1-phosphate guanylyltransferase, VTC2 (EC2.7.n.n), an L-galactose 1-phosphate phosphatase, VTC4 (EC 3.1.3.n), anL-galactose dehydrogenase, At4G33670 (EC 1.1.1.122), and anL-galactonolactone oxidase, ATGLDH (EC 1.3.3.12).

In another embodiment, the engineered cell provided by the inventioncomprises an engineered nucleic acid encoding an activity selected fromthe group consisting of a C-16:1 thioesterase, fatB, a malonyl-CoA:ACPtransacylase, fabD, an alcohol reductase, acrl, a decarbonylase, cer1,and a gene listed in Table 11.

In yet another embodiment, the engineered cell provided by the inventioncomprises an engineered nucleic acid encoding a gene selected from thegroup consisting of E. coli tesA; E. coli fadD; and A. baylyi wax-dgat.

In certain embodiments, the engineered cell provided by the invention iscapable of producing an alkane, alkene, methyl ester or ethyl ester.

In certain other embodiments, the engineered cell provided by theinvention comprises an engineered nucleic acid encoding an MEV pathwayenzyme. In some embodiments, the MEV pathway enzyme is selected from thegroup consisting of acetyl CoA thiolase, an HMG CoA synthase, an HMG CoAreductase, a mevalonate kinase, a phosphomevalonate kinase, a mevalonatepyrophosphate decarboxylase, and an IPP isomerase.

In yet another embodiment, the engineered cell provided by the inventioncomprises an engineered nucleic acid encoding a DXP pathway enzyme. Inrelated embodiments, the DXP pathway enzyme is selected from the groupconsisting of a 1-deoxy-D-xylulose 5-phosphate synthase, a1-deoxy-D-xylulose 5-phosphate reductoisomerase, a4-diphosphocytidyl-2C-methyl-D-erythritol synthase, a 4-diphosphocytidyl2C-methyl-D-erythritol kinase, a 2C methyl D erythritol 2, 4cyclodiphosphate synthase, a 1 hydroxy 2 methyl 2 (E) butenyl 4diphosphate synthase, and an isopentyl/dimethylallyl diphosphatesynthase.

In yet another embodiment, the engineered cell provided by the inventioncomprises a nucleic acid encoding an activity selected from the groupconsisting of a homocitrate synthase, lys21 (EC 2.3.3.14), ahomoaconitase, lys4, lys3 (EC 4.2.1.36), a homoisocitrate dehydrogenase,lys12, lys11, lys10 (EC 1.1.1.87), a 2-aminoadipate transaminase, aro8(EC 2.6.1.39), a phosphoglycerate dehydrogenase, serA (EC 1.1.1.95), aphosphoserine transaminase serC (EC 2.6.1.52), a phosphoserinephosphatase, serB (EC 3.1.3.3), a serine O acetyltransferase, AtSerat2;1 (EC 2.3.1.30), a cysteine synthase, At1G55880 (EC 2.5.1.47), anacetolactate synthase, ilvN, ilvB (EC 2.2.1.6), an acetohydroxyacidisomeroreductase, ilvC (EC 1.1.1.86), a dihydroxyacid dehydratase, ilvD(EC 4.2.1.9), a valine transaminase, ilvE (EC 2.6.1.42), an ACVsynthetase, Ava_(—)1613 (EC 6.3.2.26), an isopenicillin N synthase,Ava_(—)5009 (EC 1.21.3.1), converts N [L 5 amino 5 carboxypentanoyl]Lcysteinyl D valine and O₂ to isopenicillin N, an isopenicillin Nepimerase, cefD (EC 5.1.1.17), a cephalosporin biosynthesisexpandase/hydroxylase, cefEF (EC 1.14.20.1, 1.14.11.26), and adeacetylcephalosporin C acetyltransferase, cefG (EC 2.3.1.175).

In yet another embodiment, the engineered cell provided by the inventioncomprises a nucleic acid encoding alcohol dehydratase activity (EC4.2.1.n). In a related embodiment, the alcohol dehydratase activity isselected from the group of alcohol dehydratases having EC numbers4.2.1.2, 4.2.1.3, 4.2.1.4, 4.2.1.11, 4.2.1.17, 4.2.1.55, 4.2.1.33,4.2.1.34, 4.2.1.35, 4.2.1.54, 4.2.1.58, 4.2.1.60, 4.2.1.68, 4.2.1.74, or4.2.1.79. In yet another embodiment, the alcohol dehydratase is EC4.2.1.54.

In still other embodiments, the engineered cell provided by theinvention is capable of producing a pharmaceutical or an intermediatethereof. In certain related embodiments, the pharmaceutical orintermediate thereof is produced in an amount greater than about 1 mg,100 mg, 500 mg, 1 g, 5 g, 10 g, 20 g, 25 g, 30 g, 35 g, 40 g, 50 g, 100g, 120 g, or 150 g per liter of fermentation medium.

In still further embodiments, the engineered cell provided by theinvention is capable of producing a carbon-based product of interestcomprises a compound selected from the group consisting of an alcohol,ethanol, propanol, isopropanol, butanol, fatty alcohols, fatty acidester, wax ester, ethyl ester, methyl ester, hydrocarbons, n-alkanes,propane, octane, diesel, JP8, polymers, terephthalate, polyol,1,3-propanediol, 1,4-butanediol, PHA, PHB, acrylate, adipic acid,ε-caprolactone, isoprene, caprolactam, rubber, lactate, DHA,3-hydroxypropionate, γ-valerolactone, lysine, serine, aspartate,aspartic acid, sorbitol, ascorbate, ascorbic acid, isopentenol,lanosterol, omega-3 DHA, lycopene, itaconate, 1,3-butadiene, ethylene,propylene, succinate, citrate, citric acid, glutamate, malate, HPA,lactic acid, THF, gamma butyrolactone, pyrrolidones, hydroxybutyrate,glutamic acid, levulinic acid, acrylic acid, malonic acid, carotenoid,isoprenoid, itaconic acid, limonene, pharmaceutical or pharmaceuticalintermediates, erythromycin 7-ADCA/cephalosporin, polyketides, statin,paclitaxel, docetaxel, terpene, peptide, steroid, and an omega fattyacid. In certain embodiments, the isoprenoid produced by the cell isselected from the group consisting of an isoprenoid selected fromisopentylpyrophosphate (IPP), dimethylallyl pyrophosphate (DMAP), amonoterpene a sesquiterpene, a diterpene, a triterpene, a tetraterpene,and a polyterpene.

In additional embodiments, the engineered cell provided by the inventioncomprises an the cell is selected from eukaryotic plants, algae,cyanobacteria, green-sulfur bacteria, green non-sulfur bacteria, purplesulfur bacteria, purple non-sulfur bacteria, extremophiles, yeast,fungi, engineered organisms thereof, and synthetic organisms. In certainrelated embodiments, the cell is light dependent or fixes carbon. Inother related embodiments, the cell has autotrophic activity orphotoautotrophic activity. In other embodiments, the cell isphotoautotrophic in the presence of light and heterotrophic ormixotrophic in the absence of light. In other related embodiments, theengineered cell is a plant cell selected from the group consisting ofArabidopsis, Beta, Glycine, Jatropha, Miscanthus, Panicum, Phalaris,Populus, Saccharum, Salix, Simmondsia and Zea. In still other relatedembodiments, the engineered cell of the invention is an algae and/orcyanobacterial organism selected from the group consisting ofAcanthoceras, Acanthococcus, Acaryochloris, Achnanthes, Achnanthidium,Actinastrum, Actinochloris, Actinocyclus, Actinotaenium, Amphichrysis,Amphidinium, Amphikrikos, Amphipleura, Amphiprora, Amphithrix, Amphora,Anabaena, Anabaenopsis, Aneumastus, Ankistrodesmus, Ankyra, Anomoeoneis,Apatococcus, Aphanizomenon, Aphanocapsa, Aphanochaete, Aphanothece,Apiocystis, Apistonema, Arthrodesmus, Artherospira, Ascochloris,Asterionella, Asterococcus, Audouinella, Aulacoseira, Bacillaria,Balbiania, Bambusina, Bangia, Basichlamys, Batrachospermum, Binuclearia,Bitrichia, Blidingia, Botrdiopsis, Botrydium, Botryococcus,Botryosphaerella, Brachiomonas, Brachysira, Brachytrichia, Brebissonia,Bulbochaete, Bumilleria, Bumilleriopsis, Caloneis, Calothrix,Campylodiscus, Capsosiphon, Carteria, Catena, Cavinula, Centritractus,Centronella, Ceratium, Chaetoceros, Chaetochloris, Chaetomorpha,Chaetonella, Chaetonema, Chaetopeltis, Chaetophora, Chaetosphaeridium,Chamaesiphon, Chara, Characiochloris, Characiopsis, Characium, Charales,Chilomonas, Chlainomonas, Chlamydoblepharis, Chlamydocapsa,Chlamydomonas, Chlamydomonopsis, Chlamydomyxa, Chlamydonephris,Chlorangiella, Chlorangiopsis, Chlorella, Chlorobotrys, Chlorobrachis,Chlorochytrium, Chlorococcum, Chlorogloea, Chlorogloeopsis,Chlorogonium, Chlorolobion, Chloromonas, Chlorophysema, Chlorophyta,Chlorosaccus, Chlorosarcina, Choricystis, Chromophyton, Chromulina,Chroococcidiopsis, Chroococcus, Chroodactylon, Chroomonas, Chroothece,Chrysamoeba, Chrysapsis, Chrysidiastrum, Chrysocapsa, Chrysocapsella,Chrysochaete, Chrysochromulina, Chrysococcus, Chrysocrinus,Chrysolepidomonas, Chrysolykos, Chrysonebula, Chrysophyta, Chrysopyxis,Chrysosaccus, Chrysophaerella, Chrysostephanosphaera, Clodophora,Clastidium, Closteriopsis, Closterium, Coccomyxa, Cocconeis,Coelastrella, Coelastrum, Coelosphaerium, Coenochloris, Coenococcus,Coenocystis, Colacium, Coleochaete, Collodictyon, Compsogonopsis,Compsopogon, Conjugatophyta, Conochaete, Coronastrum, Cosmarium,Cosmioneis, Cosmocladium, Crateriportula, Craticula, Crinalium,Crucigenia, Crucigeniella, Cryptoaulax, Cryptomonas, Cryptophyta,Ctenophora, Cyanodictyon, Cyanonephron, Cyanophora, Cyanophyta,Cyanothece, Cyanothomonas, Cyclonexis, Cyclostephanos, Cyclotella,Cylindrocapsa, Cylindrocystis, Cylindrospermum, Cylindrotheca,Cymatopleura, Cymbella, Cymbellonitzschia, Cystodinium Dactylococcopsis,Debarya, Denticula, Dermatochrysis, Dermocarpa, Dermocarpella,Desmatractum, Desmidium, Desmococcus, Desmonema, Desmosiphon,Diacanthos, Diacronema, Diadesmis, Diatoma, Diatomella, Dicellula,Dichothrix, Dichotomococcus, Dicranochaete, Dictyochloris, Dictyococcus,Dictyosphaerium, Didymocystis, Didymogenes, Didymosphenia, Dilabifilum,Dimorphococcus, Dinobryon, Dinococcus, Diplochloris, Diploneis,Diplostauron, Distrionella, Docidium, Draparnaldia, Dunaliella,Dysmorphococcus, Ecballocystis, Elakatothrix, Ellerbeckia, Encyonema,Enteromorpha, Entocladia, Entomoneis, Entophysalis, Epichrysis,Epipyxis, Epithemia, Eremosphaera, Euastropsis, Euastrum, Eucapsis,Eucocconeis, Eudorina, Euglena, Euglenophyta, Eunotia, Eustigmatophyta,Eutreptia, Fallacia, Fischerella, Fragilaria, Fragilariforma, Franceia,Frustulia, Curcilla, Geminella, Genicularia, Glaucocystis, Glaucophyta,Glenodiniopsis, Glenodinium, Gloeocapsa, Gloeochaete, Gloeochrysis,Gloeococcus, Gloeocystis, Gloeodendron, Gloeomonas, Gloeoplax,Gloeothece, Gloeotila, Gloeotrichia, Gloiodictyon, Golenkinia,Golenkiniopsis, Gomontia, Gomphocymbella, Gomphonema, Gomphosphaeria,Gonatozygon, Gongrosia, Gongrosira, Goniochloris, Gonium, Gonyostomum,Granulochloris, Granulocystopsis, Groenbladia, Gymnodinium, Gymnozyga,Gyrosigma, Haematococcus, Hafniomonas, Hallassia, Hammatoidea, Hannaea,Hantzschia, Hapalosiphon, Haplotaenium, Haptophyta, Haslea, Hemidinium,Hemitoma, Heribaudiella, Heteromastix, Heterothrix, Hibberdia,Hildenbrandia, Hillea, Holopedium, Homoeothrix, Hormanthonema,Hormotila, Hyalobrachion, Hyalocardium, Hyalodiscus, Hyalogonium,Hyalotheca, Hydrianum, Hydrococcus, Hydrocoleum, Hydrocoryne,Hydrodictyon, Hydrosera, Hydrurus, Hyella, Hymenomonas, Isthmochloron,Johannesbaptistia, Juranyiella, Karayevia, Kathablepharis, Katodinium,Kephyrion, Keratococcus, Kirchneriella, Klebsormidium, Kolbesia,Koliella, Komarekia, Korshikoviella, Kraskella, Lagerheimia, Lagynion,Lamprothamnium, Lemanea, Lepocinclis, Leptosira, Lobococcus, Lobocystis,Lobomonas, Luticola, Lyngbya, Malleochloris, Mallomonas, Mantoniella,Marssoniella, Martyana, Mastigocoleus, Gastogloia, Melosira,Merismopedia, Mesostigma, Mesotaenium, Micractinium, Micrasterias,Microchaete, Microcoleus, Microcystis, Microglena, Micromonas,Microspora, Microthamnion, Mischococcus, Monochrysis, Monodus,Monomastix, Monoraphidium, Monostroma, Mougeotia, Mougeotiopsis,Myochloris, Myromecia, Myxosarcina, Naegeliella, Nannochloris,Nautococcus, Navicula, Neglectella, Neidium, Nephroclamys, Nephrocytium,Nephrodiella, Nephroselmis, Netrium, Nitella, Nitellopsis, Nitzschia,Nodularia, Nostoc, Ochromonas, Oedogonium, Oligochaetophora, Onychonema,Oocardium, Oocystis, Opephora, Ophiocytium, Orthoseira, Oscillatoria,Oxyneis, Pachycladella, Palmella, Palmodictyon, Pnadorina, Pannus,Paralia, Pascherina, Paulschulzia, Pediastrum, Pedinella, Pedinomonas,Pedinopera, Pelagodictyon, Penium, Peranema, Peridiniopsis, Peridinium,Peronia, Petroneis, Phacotus, Phacus, Phaeaster, Phaeodermatium,Phaeophyta, Phaeosphaera, Phaeothamnion, Phormidium, Phycopeltis,Phyllariochloris, Phyllocardium, Phyllomitas, Pinnularia, Pitophora,Placoneis, Planctonema, Planktosphaeria, Planothidium, Plectonema,Pleodorina, Pleurastrum, Pleurocapsa, Pleurocladia, Pleurodiscus,Pleurosigma, Pleurosira, Pleurotaenium, Pocillomonas, Podohedra,Polyblepharides, Polychaetophora, Polyedriella, Polyedriopsis,Polygoniochloris, Polyepidomonas, Polytaenia, Polytoma, Polytomella,Porphyridium, Posteriochromonas, Prasinochloris, Prasinocladus,Prasinophyta, Prasiola, Prochlorphyta, Prochlorothrix, Protoderma,Protosiphon, Provasoliella, Prymnesium, Psammodictyon, Psammothidium,Pseudanabaena, Pseudenoclonium, Psuedocarteria, Pseudochate,Pseudocharacium, Pseudococcomyxa, Pseudodictyosphaerium,Pseudokephyrion, Pseudoncobyrsa, Pseudoquadrigula, Pseudosphaerocystis,Pseudostaurastrum, Pseudostaurosira, Pseudotetrastrum, Pteromonas,Punctastruata, Pyramichlamys, Pyramimonas, Pyrrophyta, Quadrichloris,Quadricoccus, Quadrigula, Radiococcus, Radiofilum, Raphidiopsis,Raphidocelis, Raphidonema, Raphidophyta, Peimeria, Rhabdoderma,Rhabdomonas, Rhizoclonium, Rhodomonas, Rhodophyta, Rhoicosphenia,Rhopalodia, Rivularia, Rosenvingiella, Rossithidium, Roya, Scenedesmus,Scherffelia, Schizochlamydella, Schizochlamys, Schizomeris, Schizothrix,Schroederia, Scolioneis, Scotiella, Scotiellopsis, Scourfieldia,Scytonema, Selenastrum, Selenochloris, Sellaphora, Semiorbis,Siderocelis, Diderocystopsis, Dimonsenia, Siphononema, Sirocladium,Sirogonium, Skeletonema, Sorastrum, Spermatozopsis, Sphaerellocystis,Sphaerellopsis, Sphaerodinium, Sphaeroplea, Sphaerozosma,Spiniferomonas, Spirogyra, Spirotaenia, Spirulina, Spondylomorum,Spondylosium, Sporotetras, Spumella, Staurastrum, Stauerodesmus,Stauroneis, Staurosira, Staurosirella, Stenopterobia, Stephanocostis,Stephanodiscus, Stephanoporos, Stephanosphaera, Stichococcus,Stichogloea, Stigeoclonium, Stigonema, Stipitococcus, Stokesiella,Strombomonas, Stylochrysalis, Stylodinium, Styloyxis, Stylosphaeridium,Surirella, Sykidion, Symploca, Synechococcus, Synechocystis, Synedra,Synochromonas, Synura, Tabellaria, Tabularia, Teilingia, Temnogametum,Tetmemorus, Tetrachlorella, Tetracyclus, Tetradesmus, Tetraedriella,Tetraedron, Tetraselmis, Tetraspora, Tetrastrum, Thalassiosira,Thamniochaete, Thermosynechococcus, Thorakochloris, Thorea, Tolypella,Tolypothrix, Trachelomonas, Trachydiscus, Trebouxia, Trentepholia,Treubaria, Tribonema, Trichodesmium, Trichodiscus, Trochiscia,Tryblionella, Ulothrix, Uroglena, Uronema, Urosolenia, Urospora, Uva,Vacuolaria, Vaucheria, Volvox, Volvulina, Westella, Woloszynskia,Xanthidium, Xanthophyta, Xenococcus, Zygnema, Zygnemopsis, and Zygonium.In yet other related embodiments, the engineered cell provided by theinvention is derived from a Chloroflexus, Chloronema, Oscillochloris,Heliothrix, Herpetosiphon, Roseiflexus, and Thermomicrobium cell; agreen sulfur bacteria selected from: Chlorobium, Clathrochloris, andProsthecochloris; a purple sulfur bacteria is selected from:Allochromatium, Chromatium, Halochromatium, Isochromatium,Marichromatium, Rhodovulum, Thermochromatium, Thiocapsa,Thiorhodococcus, and Thiocystis; a purple non-sulfur bacteria isselected from: Phaeospirillum, Rhodobaca, Rhodobacter, Rhodomicrobium,Rhodopila, Rhodopseudomonas, Rhodothalassium, Rhodospirillum,Rodovibrio, and Roseospira; an aerobic chemolithotrophic bacteriaselected from: nitrifying bacteria. Nitrobacteraceae sp., Nitrobactersp., Nitrospina sp., Nitrococcus sp., Nitrospira sp., Nitrosomonas sp.,Nitrosococcus sp., Nitrosospira sp., Nitrosolobus sp., Nitrosovibriosp.; colorless sulfur bacteria such as, Thiovulum sp., Thiobacillus sp.,Thiomicrospira sp., Thiosphaera sp., Thermothrix sp.; obligatelychemolithotrophic hydrogen bacteria, Hydrogenobacter sp., iron andmanganese-oxidizing and/or depositing bacteria, Siderococcus sp., andmagnetotactic bacteria, Aquaspirillum sp; an archaeobacteria selectedfrom: methanogenic archaeobacteria, Methanobacterium sp.,Methanobrevibacter sp., Methanothermus sp., Methanococcus sp.,Methanomicrobium sp., Methanospirillum sp., Methanogenium sp.,Methanosarcina sp., Methanolobus sp., Methanothrix sp., Methanococcoidessp., Methanoplanus sp.; extremely thermophilic sulfur-Metabolizers suchas Thermoproteus sp., Pyrodictium sp., Sulfolobus sp., Acidianus sp.,Bacillus subtilis, Saccharomyces cerevisiae, Streptomyces sp., Ralstoniasp., Rhodococcus sp., Corynebacteria sp., Brevibacteria sp.,Mycobacteria sp., and oleaginous yeast; and extremophile selected fromPyrolobusfumarii; Synechococcus lividis, mesophiles, psychrophiles,Psychrobacter, insects, Deinococcus radiodurans, piezophiles,barophiles, hypergravity tolerant organisms, hypogravity tolerantorganisms, vacuum tolerant organisms, tardigrades, insects, microbesseeds, dessicant tolerant anhydrobiotic organisms, xerophiles, Artemiasalina, nematodes, microbes, fungi, lichens, salt tolerant organismshalophiles, halobacteriacea, Dunaliella salina, pH tolerant organisms,alkaliphiles, Natronobacterium, Bacillus firmus OF4, Spirulina spp.,acidophiles, Cyanidium caldarium, Ferroplasma sp., anaerobes, whichcannot tolerate O₂ , Methanococcus jannaschii, microaerophils, whichtolerate some O₂ , Clostridium, aerobes, which require O₂, gas tolerantorganisms, which tolerate pure CO₂ , Cyanidium caldarium, metal tolerantorganisms, metalotolerants, Ferroplasma acidarmanus Ralstonia sp. CH34.

In yet other embodiments, engineered cell provided by the invention isderived from Arabidopsis thaliana, Panicum virgatum, Miscanthusgiganteus, and Zea mays (plants), Botryococcus braunii, Chlamydomonasreinhardtii and Dunaliela salina (algae), Synechococcus sp. PCC 7002,Synechococcus sp. PCC 7942, Synechocystis sp. PCC 6803, andThermosynechococcus elongatus BP-1 (cyanobacteria), Chlorobium tepidum(green sulfur bacteria), Chloroflexus auranticus (green non-sulfurbacteria), Chromatium tepidum and Chromatium vinosum (purple sulfurbacteria), Rhodospirillum rubrum, Rhodobacter capsulatus, andRhodopseudomonas palusris (purple non-sulfur bacteria).

In still other embodiments, the engineered cell provided by theinvention is a Clostridium ljungdahlii, Clostridium thermocellum,Penicillium chrysogenum, Pichia pastoris, Saccharomyces cerevisiae,Schizosaccharomyces pombe, Pseudomonasfluorescens, or Zymomonas mobiliscell.

In certain embodiments, the engineered cell provided by the inventionare capable of conducting or regulating at least one metabolic pathwayselected from the group consisting of photosynthesis, sulfate reduction,methanogenesis, acetogenesis, reductive TCA cycle, Calvin cycle, 3-HPAcycle and 3HP/4HB cycle.

In certain other embodiments, the invention provides a method forproducing carbon-based product of interest or intermediate thereofcomprising: introducing one or more engineered nucleic acids into acarbon fixing organism, wherein said engineered host cell is capable ofusing a minimum amount of light energy to synthesize a carbon-basedproduct of interest directly from carbon dioxide and water, culturingsaid engineered host cell, then isolating a carbon-based product ofinterest from said engineered cell or culture medium. In a relatedembodiment, the cell is cultured in a photobioreactor. In anotherrelated embodiment, the carbon-based products of interest are released,permeated or exported from the cell. In yet another related embodiment,the carbon-based product is isolated from the culture medium.

In certain embodiments, the engineered cell provided by the invention iscapable of producing a carbon-based product of interest characterized ashaving −δ_(p)(‰) of about 63.5 to about 66 and −D(‰) of about 37.5 toabout 40.

The invention also provides a method for monetizing carbon-based productof interest production and CO₂ taken up in association with theproduction of a carbon-based product of interest by an engineeredphotoautotrophic organism, comprising: quantifying an amount of saidproduct produced by said engineered photoautotrophic organism;quantifying an amount of CO₂ taken up in association with said amount ofsaid product produced; valuing said amount of said product according toa market value; valuing said amount of CO₂ taken up in association withsaid amount of product produced; and monetizing said amounts of saidproduct produced and CO₂ taken up in association with said amount ofsaid product produced by selling said amount of said product and sellinga CO₂ credit into a carbon market.

The invention also provides a method for the biogenic production ofethanol, comprising: culturing an engineered cyanobacterium in a culturemedium in the presence of light and CO2, wherein said cyanobacteriumcomprises at least two copies of a recombinant alcohol dehydrogenasegene, and wherein at least one copy of said recombinant alcoholdehydrogenase gene is extrachromosomal. In certain related embodiments,the expression of at least one copy of said recombinant alcoholdehydrogenase gene is driven by a lambda cI promoter.

In another embodiment, the invention provides a method for increasingthe production of ethanol by an engineered cyanobacterium, comprisingthe step of varying the activity of recombinantly expressed alcoholdehydrogenase and pyruvate decarboxylase in said cyanobacterium. Incertain embodiments, the cyanobacterium is a thermophile. In a relatedembodiment, the activity levels are varied by differentially expressingthe enzymes alcohol dehydrogenase and pyruvate decarboxylase. In yetanother embodiment, the differential expression is achieved bymodulating the strength of a promoter that controls expression ofalcohol dehydrogenase or pyruvate decarboxylase. In yet anotherembodiment, the method of claim 4, wherein said expression of alcoholdehydrogenase is driven by promoters on at least two distinct plasmids,e.g., a plasmid selected from the group of plasmids consisting of AQ1,pAQ3, pAQ4, pAQ5, pAQ6, and pAQ7. In yet another embodiment, theactivity is varied by controlling the level of a co-factor required byalcohol dehydrogenase or pyruvate decarboxylase.

In certain embodiments of the method for increasing the production ofethanol by an engineered cyanobacterium, the measured level ofacetaldehyde released into said culture medium by said engineeredorganism is less than about 7 mg/ml after approximately 10 days ofculture. In certain other embodiments, the cumulative amount of ethanolreleased into said culture medium by said engineered organism is equalto or greater than about 4 g/L after approximately 380 hours. In yetother embodiments, the measured level of ethanol released into saidculture medium by said engineered organism is at least about 1750mgs/ml. In still other embodiments, the measured concentration (mg/ml)of ethanol released into said culture medium by said engineered organismis 100, 200 or 300 fold higher than the measured level of acetaldehydein said culture medium.

The invention also provides an engineered cyanobacterium comprisingalcohol dehydrogenase under the control of the lambda cI promoter. Inrelated embodiments, the engineered cyanobacterium is an engineeredSynechococcus strain comprising at least two engineered plasmids. Forexample, the plasmics could be selected from the group consisting ofpAQ1, pAQ3, pAQ4, pAQ5, pAQ6, and pAQ7. In related embodiments, the twoengineered plasmids separately encode a recombinant alcoholdehydrogenase activity. In yet other related embodiments, therecombinant alcohol dehydrogenase activity is adhA_(M). In oneembodiment, the cyanobacterium lacks a functioning lactate dehydrogenasegene or lactate dehydrogenase enzyme activity. In yet anotherembodiment, the cyanobacterium is engineered to express a recombinantpyruvate decarboxylase activity in addition to the alcohol dehydrogenaseactivity. In yet another embodiment, the recombinant pyruvatedecarboxylase activity is encoded by one of the least two engineeredplasmids encoding the recombinant alcohol dehydrogenase activity.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1(A-O) provides various genes identified that can be expressed,upregulated, attenuated or knocked out in engineering carbon dioxidefixing microorganisms of the invention in the production of carbon-basedproducts of interest.

FIG. 2 provides an example of pathways to produce ethanol, succinate andother derivates.

FIG. 3 provides a schematic diagram to produce ethylene from GAP.

FIG. 4 provides an example of a pathway for n-alkane and fatty alcoholsynthesis.

FIG. 5 provides an example of pathways to produce several differentchemicals: succinate, glutamate, itaconic acid and 3-hydroypropionate.

FIG. 6 provides a schematic to convert succinate or 3-hydroxypropionateto various chemicals.

FIG. 7 provides a schematic of glutamate or itaconic acid conversion tovarious chemicals.

FIG. 8 provides a schematic of pathways to produce butanediol fromsuccinate.

FIG. 9 shows the GC/FID chromatogram of the control Synechococcus 7002strain.

FIG. 10 shows the GC/FID chromatogram of the 7002/efe_rs recombinantstrain.

FIG. 11 graphically illustrates optical density of select ethanologensover time.

FIG. 12 graphically illustrates ethanol concentrations of cultures inthe supernatant over time.

FIG. 13 graphically illustrates acetaldehdye concentrations of culturesin the supernatant over time.

FIG. 14 graphically illustrates ethanol to acetaldehyde ratios ofcultures in the supernatant over time.

FIG. 15 graphically illustrates ratios of ethanol concentration to OD(730 nm) of cultures in the supernatant over time.

FIG. 16 depicts a total ion chromatograph for JCC342c, JCC545, andJCC547 in the retention time window during which TMS-derivatizedα-D-glucose and β-D-glucose elute. The JCC543 trace has been omitted forclarity.

FIG. 17 depicts a total ion chromatograph for JCC342c, JCC545, andJCC546 in the retention time window during which TMS-derivatized sucroseelute. Cell densities were as indicated in FIG. 16. The JCC543 trace hasbeen omitted for clarity.

FIG. 18 shows representative GC/MS chromatograms of JCC738 (top trace)and JCC724 (bottom trace) analyzed for the presence of maltose. Thepeaks for α-maltose and β-maltose are indicated.

FIG. 19 is a table indicating the amounts of maltose found in cellpellet extracts given as mg/L of culture.

FIG. 20 depicts representative GC/FID chromatograms of strains analyzedfor the presence of ethyl esters. The peaks for ethyl myristate, ethylpalmitate, ethyl oleate and ethyl stearate are indicated. (A) JCC879(resistance marker) 236 h timepoint flask #1; (B) JCC750 (PaphII-tesA)236 h timepoint flask #1; (C) JCC803 (lacIq Ptrc-tesA-fadD-wax) 236 htimepoint flask #1; (D) JCC723 (PaphII-tesA-fadD-wax) 236 h timepointflask #1.

FIG. 21 depicts a GC/MS chromatogram overlay comparing cell pelletextracts of JCC803 incubated with either methanol (top trace) or ethanol(bottom traces). The peaks due to methyl esters (MEs) or ethyl esters(EEs) are labeled.

DETAILED DESCRIPTION Abbreviations and Terms

The following explanations of terms and methods are provided to betterdescribe the present disclosure and to guide those of ordinary skill inthe art in the practice of the present disclosure. As used herein,“comprising” means “including” and the singular forms “a” or “an” or“the” include plural references unless the context clearly dictatesotherwise. For example, reference to “comprising a cell” includes one ora plurality of such cells, and reference to “comprising thethioesterase” includes reference to one or more thioesterase peptidesand equivalents thereof known to those of ordinary skill in the art, andso forth. The term “or” refers to a single element of stated alternativeelements or a combination of two or more elements, unless the contextclearly indicates otherwise.

Unless explained otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this disclosure belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. The materials, methods, and examples areillustrative only and not intended to be limiting. Other features of thedisclosure are apparent from the following detailed description and theclaims.

Accession Numbers: The accession numbers throughout this description arederived from the NCBI database (National Center for BiotechnologyInformation) maintained by the National Institute of Health, U.S.A. Theaccession numbers are as provided in the database on Feb. 1, 2008.

Enzyme Classification Numbers (EC): The EC numbers provided throughoutthis description are derived from the KEGG Ligand database, maintainedby the Kyoto Encyclopedia of Genes and Genomics, sponsored in part bythe University of Tokyo. The EC numbers are as provided in the databaseon Feb. 1, 2008.

Amino acid: Triplets of nucleotides, referred to as codons, in DNAmolecules code for amino acid in a peptide. The term codon is also usedfor the corresponding (and complementary) sequences of three nucleotidesin the mRNA into which the DNA sequence is transcribed.

Attenuate: The term as used herein generally refers to a functionaldeletion, including a mutation, partial or complete deletion, insertion,or other variation made to a gene sequence or a sequence controlling thetranscription of a gene sequence, which reduces or inhibits productionof the gene product, or renders the gene product non-functional. In someinstances a functional deletion is described as a knockout mutation.Attenuation also includes amino acid sequence changes by altering thenucleic acid sequence, placing the gene under the control of a lessactive promoter, downregulation, expressing interfering RNA, ribozymesor antisense sequences that target the gene of interest, or through anyother technique known in the art. In one example, the sensitivity of aparticular enzyme to feedback inhibition or inhibition caused by acomposition that is not a product or a reactant (non-pathway specificfeedback) is lessened such that the enzyme activity is not impacted bythe presence of a compound. In other instances, an enzyme that has beenaltered to be less active can be referred to as attenuated.

“Carbon-based Products of Interest” include alcohols such as ethanol,propanol, isopropanol, butanol, fatty alcohols, fatty acid esters, waxesters; hydrocarbons and alkanes such as propane, octane, diesel, JetPropellant 8, polymers such as terephthalate, 1,3-propanediol,1,4-butanediol, polyols, polyhydroxyalkanoates (PHAs),polyhydroxybutyrates (PHBs), acrylate, adipic acid, ε-caprolactone,isoprene, caprolactam, rubber; commodity chemicals such as lactate,docosahexaenoic acid (DHA), 3-hydroxypropionate, γ-valerolactone,lysine, serine, aspartate, aspartic acid, sorbitol, ascorbate, ascorbicacid, isopentenol, lanosterol, omega-3 DHA, lycopene, itaconate,1,3-butadiene, ethylene, propylene, succinate, citrate, citric acid,glutamate, malate, 3-hydroxyprionic acid (HPA), lactic acid, THF, gammabutyrolactone, pyrrolidones, hydroxybutyrate, glutamic acid, levulinicacid, acrylic acid, malonic acid; specialty chemicals such ascarotenoids, isoprenoids, itaconic acid; pharmaceuticals andpharmaceutical intermediates such as 7-aminodesacetoxycephalosporonicacid, cephalosporin, erythromycin, polyketides, statins, paclitaxel,docetaxel, terpenes, peptides, steroids, omega fatty acids and othersuch suitable products of interest. Such products are useful in thecontext of biofuels, industrial and specialty chemicals, asintermediates used to make additional products, such as nutritionalsupplements, neutraceuticals, polymers, paraffin replacements, personalcare products and pharmaceuticals.

Deletion: The removal of one or more nucleotides from a nucleic acidmolecule or one or more amino acids from a protein, the regions oneither side being joined together.

DNA: Deoxyribonucleic acid. DNA is a long chain polymer which includesthe genetic material of most living organisms (some viruses have genesincluding ribonucleic acid, RNA). The repeating units in DNA polymersare four different nucleotides, each of which includes one of the fourbases, adenine, guanine, cytosine and thymine bound to a deoxyribosesugar to which a phosphate group is attached.

Endogenous: As used herein with reference to a nucleic acid molecule anda particular cell or microorganism refers to a nucleic acid sequence orpeptide that is in the cell and was not introduced into the cell (or itsprogentors) using recombinant engineering techniques. For example, agene that was present in the cell when the cell was originally isolatedfrom nature. A gene is still considered endogenous if the controlsequences, such as a promoter or enhancer sequences that activatetranscription or translation have been altered through recombinanttechniques.

“An enzyme activity”: As used herein, the term “an enzyme activity”means that the indicated enzyme (e.g., “an alcohol dehydrogenaseactivity”) has measurable attributes in terms of, e.g., substratespecific activity, pH and temperature optima, and other standardmeasures of enzyme activity as the activity encoded by a referenceenzyme (e.g., alcohol dehydrogenase). Furthermore, the enzyme is atleast 90% identical at a nucleic or amino acid level to the sequence ofthe reference enzyme as measured by a BLAST search.

Exogenous: As used herein with reference to a nucleic acid molecule anda particular cell or microorganism refers to a nucleic acid sequence orpeptide that was not present in the cell when the cell was originallyisolated from nature. For example, a nucleic acid that originated in adifferent microorganism and was engineered into an alternate cell usingrecombinant DNA techniques or other methods for delivering said nucleicacid is exogenous.

Expression: The process by which a gene's coded information is convertedinto the structures and functions of a cell, such as a protein, transferRNA, or ribosomal RNA. Expressed genes include those that aretranscribed into mRNA and then translated into protein and those thatare transcribed into RNA but not translated into protein (for example,transfer and ribosomal RNAs).

Expression Control Sequence: as used herein refers to polynucleotidesequences which are necessary to affect the expression of codingsequences to which they are operatively linked. Expression controlsequences are sequences which control the transcription,post-transcriptional events and translation of nucleic acid sequences.Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (e.g., ribosome binding sites); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein secretion. The nature of such control sequences differsdepending upon the host organism; in prokaryotes, such control sequencesgenerally include promoter, ribosomal binding site, and transcriptiontermination sequence. The term “control sequences” is intended toinclude, at a minimum, all components whose presence is essential forexpression, and can also include additional components whose presence isadvantageous, for example, leader sequences and fusion partnersequences.

Overexpression: When a gene is caused to be transcribed at an elevatedrate compared to the endogenous transcription rate for that gene. Insome examples, overexpression additionally includes an elevated rate oftranslation of the gene compared to the endogenous translation rate forthat gene. Methods of testing for overexpression are well known in theart, for example transcribed RNA levels can be assessed using reversetranscriptase polymerase chain reaction (RT-PCR) and protein levels canbe assessed using sodium dodecyl sulfate polyacrylamide gelelecrophoresis (SDS-PAGE) analysis. Furthermore, a gene is considered tobe overexpressed when it exhibits elevated activity compared to itsendogenous activity, which may occur, for example, through reduction inconcentration or activity of its inhibitor, or via expression of mutantversion with elevated activity. In preferred embodiments, when the hostcell encodes an endogenous gene with a desired biochemical activity, itis useful to overexpress an exogenous gene, which allows for moreexplicit regulatory control in the fermentation and a means topotentially mitigate the effects of central metabolism regulation, whichis focused around the native genes explicity.

Downregulation: When a gene is caused to be transcribed at a reducedrate compared to the endogenous gene transcription rate for that gene.In some examples, downregulation additionally includes a reduced levelof translation of the gene compared to the endogenous translation ratefor that gene. Methods of testing for downregulation are well known tothose in the art. For example, the transcribed RNA levels can beassessed using RT-PCR, and protein levels can be assessed using SDS-PAGEanalysis.

Knock-out: A gene whose level of expression or activity has been reducedto zero. In some examples, a gene is knocked-out via deletion of some orall of its coding sequence. In other examples, a gene is knocked-out viaintroduction of one or more nucleotides into its open-reading frame,which results in translation of a non-sense or otherwise non-functionalprotein product.

Autotroph: Autotrophs (or autotrophic organisms) are organisms thatproduce complex organic compounds from simple inorganic molecules and anexternal source of energy, such as light (photoautotroph) or chemicalreactions of inorganic compounds.

Hydrocarbon: The term generally refers to a chemical compound thatconsists of the elements carbon (C), hydrogen (H) and optionally oxygen(O). There are essentially three types of hydrocarbons, e.g., aromatichydrocarbons, saturated hydrocarbons and unsaturated hydrocarbons suchas alkenes, alkynes, and dienes. The term also includes fuels, biofuels,plastics, waxes, solvents and oils. Hydrocarbons encompass biofuels, aswell as plastics, waxes, solvents and oils.

“Immiscible” or “Immiscibility” refers to the relative inability of acompound to dissolve in water and is defined by the compounds partitioncoefficient. The partition coefficient, P, is defined as the equilibriumconcentration of compound in an organic phase (in a bi-phasic system theorganic phase is usually the phase formed by the fatty acid derivativeduring the production process, however, in some examples an organicphase can be provided (such as a layer of octane to facilitate productseparation) divided by the concentration at equilibrium in an aqueousphase (i.e., fermentation broth). When describing a two phase system theP is usually discussed in terms of log P. A compound with a log P of 10would partition 10:1 to the organic phase, while a compound of log P of0.1 would partition 10:1 to the aqueous phase.

Biosynthetic pathway: Also referred to as “metabolic pathway,” refers toa set of anabolic or catabolic biochemical reactions for converting(transmuting) one chemical species into another. For example, ahydrocarbon biosynthetic pathway refers to the set of biochemicalreactions that convert inputs and/or metabolites to hydrocarbonproduct-like intermediates and then to hydrocarbons or hydrocarbonproducts. Anabolic pathways involve constructing a larger molecule fromsmaller molecules, a process requiring energy. Catabolic pathwaysinvolve breaking down of larger: molecules, often releasing energy.

Cellulose: Cellulose [(C₆H₁₀O₅)_(n)] is a long-chain polymerpolysaccharide carbohydrate, of beta-glucose. It forms the primarystructural component of plants and is not digestible by humans.Cellulose is a common material in plant cell walls and was first notedas such in 1838. It occurs naturally in almost pure form only in cottonfiber; in combination with lignin and any hemicellulose, it is found inall plant material.

Biofuel: A biofuel is any fuel that derives from a biological source.Biofuel refers to one or more hydrocarbons, one or more alcohols, one ormore fatty esters or a mixture thereof. Preferably, liquid hydrocarbonsare used.

“Fuel component” is any compound or a mixture of compounds that are usedto formulate a fuel composition. There are “major fuel components” and“minor fuel components.” A major fuel component is present in a fuelcomposition by at least 50% by volume; and a minor fuel component ispresent in a fuel composition by less than 50%. Fuel additives are minorfuel components. The isoprenoid compounds disclosed herein can be amajor component or a minor component, by themselves or in a mixture withother fuel components.

As used herein, a composition that is a “substantially pure” compound issubstantially free of one or more other compounds, i.e., the compositioncontains greater than 80 vol. %, greater than 90 vol. %, greater than 95vol. %, greater than 96 vol. %, greater than 97 vol. %, greater than 98vol. %, greater than 99 vol. %, greater than 99.5 vol. %, greater than99.6 vol. %, greater than 99.7 vol. %, greater than 99.8 vol. %, orgreater than 99.9 vol. % of the compound; or less than 20 vol. %, lessthan 10 vol. %, less than 5 vol. %, less than 3 vol. %, less than 1 vol.%, less than 0.5 vol. %, less than 0.1 vol. %, or less than 0.01 vol. %of the one or more other compounds, based on the total volume of thecomposition.

Nucleic Acid Molecule: The term encompasses both RNA and DNA moleculesincluding, without limitation, cDNA, genomic DNA, and mRNA and alsoincludes synthetic nucleic acid molecules, such as those that arechemically synthesized or recombinantly produced. The nucleic acidmolecule can be double-stranded or single-stranded, circular or linear.If single-stranded, the nucleic acid molecule can be the sense strand orthe antisense strand.

Engineered nucleic acid: An “engineered nucleic acid” is a nucleic acidmolecule that includes at least one difference from anaturally-occurring nucleic acid molecule. An engineered nucleic acidincludes all exogenous modified and unmodified heterologous sequences(i.e., sequences derived from an organism or cell other than thatharboring the engineered nucleic acid) as well as endogenous genes,operons, coding sequences, or non-coding sequences, that have beenmodified, mutated, or that include deletions or insertions as comparedto a naturally-occurring sequence. Engineered nucleic acids also includeall sequences, regardless of origin, that are linked to an induciblepromoter or to another control sequence with which they are notnaturally associated.

Suitable fermentation conditions. The term generally refers tofermentation media and conditions adjustable with, pH, temperature,levels of aeration, etc., preferably optimum conditions that allowmicroorganisms to produce carbon-based products of interest. Todetermine if culture conditions permit product production, themicroorganism can be cultured for about 24 hours to one week afterinoculation and a sample can be obtained and analyzed. The cells in thesample or the medium in which the cells are grown are tested for thepresence of the desired product.

Isolated: An “isolated” nucleic acid or polynucleotide (e.g., an RNA,DNA or a mixed polymer) is one which is substantially separated fromother cellular components that naturally accompany the nativepolynucleotide in its natural host cell, e.g., ribosomes, polymerases,and genomic sequences with which it is naturally associated. The termembraces a nucleic acid or polynucleotide that (1) has been removed fromits naturally occurring environment, (2) is not associated with all or aportion of a polynucleotide in which the “isolated polynucleotide” isfound in nature, (3) is operatively linked to a polynucleotide which itis not linked to in nature, or (4) does not occur in nature. The term“isolated” or “substantially pure” also can be used in reference torecombinant or cloned DNA isolates, chemically synthesizedpolynucleotide analogs, or polynucleotide analogs that are biologicallysynthesized by heterologous systems. However, “isolated” does notnecessarily require that the nucleic acid or polynucleotide so describedhas itself been physically removed from its native environment. Forinstance, an endogenous nucleic acid sequence in the genome of anorganism is deemed “isolated” herein if a heterologous sequence (i.e., asequence that is not naturally adjacent to this endogenous nucleic acidsequence) is placed adjacent to the endogenous nucleic acid sequence,such that the expression of this endogenous nucleic acid sequence isaltered. By way of example, a non-native promoter sequence can besubstituted (e.g. by homologous recombination) for the native promoterof a gene in the genome of a human cell, such that this gene has analtered expression pattern. This gene would now become “isolated”because it is separated from at least some of the sequences thatnaturally flank it. A nucleic acid is also considered “isolated” if itcontains any modifications that do not naturally occur to thecorresponding nucleic acid in a genome. For instance, an endogenouscoding sequence is considered “isolated” if it contains an insertion,deletion or a point mutation introduced artificially, e.g. by humanintervention. An “isolated nucleic acid” also includes a nucleic acidintegrated into a host cell chromosome at a heterologous site, as wellas a nucleic acid construct present as an episome. Moreover, an“isolated nucleic acid” can be substantially free of other cellularmaterial, or substantially free of culture medium when produced byrecombinant techniques, or substantially free of chemical precursors orother chemicals when chemically synthesized. The term also embracesnucleic acid molecules and proteins prepared by recombinant expressionin a host cell as well as chemically synthesized nucleic acid moleculesand proteins.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein coding regions, in the samereading frame. Configurations of separate genes that are transcribed intandem as a single messenger RNA are denoted as operons. Thus placinggenes in close proximity, for example in a plasmid vector, under thetranscriptional regulation of a single promoter, constitutes a syntheticoperon.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified productpreparation, is one in which the product is more concentrated than theproduct is in its environment within a cell. For example, a purified waxis one that is substantially separated from cellular components (nucleicacids, lipids, carbohydrates, and other peptides) that can accompany it.In another example, a purified wax preparation is one in which the waxis substantially free from contaminants, such as those that might bepresent following fermentation.

In one example, a fatty acid ester is purified when at least about 50%by weight of a sample is composed of the fatty acid ester, for examplewhen at least about 60%, 70%, 80%, 85%, 90%, 92%, 95%, 98%, or 99% ormore of a sample is composed of the fatty acid ester. Examples ofmethods that can be used to purify waxes, fatty alcohols, and fatty acidesters are well-known to persons of ordinary skill in the art and aredescribed below. An example of a pathway for n-alkane and fatty alcoholsynthesis is provided in FIG. 4.

Detectable: Capable of having an existence or presence ascertained usingvarious analytical methods as described throughout the description orotherwise known to a person skilled in the art.

Recombinant: A recombinant nucleic acid molecule or protein is one thathas a sequence that is not naturally occurring, has a sequence that ismade by an artificial combination of two otherwise separated segments ofsequence, or both. This artificial combination can be achieved, forexample, by chemical synthesis or by the artificial manipulation ofisolated segments of nucleic acid molecules or proteins, such as geneticengineering techniques. Recombinant is also used to describe nucleicacid molecules that have been artificially manipulated, but contain thesame regulatory sequences and coding regions that are found in theorganism from which the nucleic acid was isolated.

The term “recombinant host cell” (“expression host cell,” “expressionhost system,” “expression system,” or simply “host cell”), as usedherein, refers to a cell into which a recombinant vector has beenintroduced, e.g., a vector comprising acyl-CoA synthase. It should beunderstood that such terms are intended to refer not only to theparticular subject cell but to the progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell” as used herein. A recombinant host cell may be anisolated cell or cell line grown in culture or may be a cell whichresides in a living tissue or organism.

Release: The movement of a compound from inside a cell (intracellular)to outside a cell (extracellular). The movement can be active orpassive. When release is active it can be facilitated by one or moretransporter peptides and in some examples it can consume energy. Whenrelease is passive, it can be through diffusion through the membrane andcan be facilitated by continually collecting the desired compound fromthe extracellular environment, thus promoting further diffusion. Releaseof a compound can also be accomplished by lysing a cell.

Surfactants: Substances capable of reducing the surface tension of aliquid in which they are dissolved. They are typically composed of awater-soluble head and a hydrocarbon chain or tail. The water solublegroup is hydrophilic and can be either ionic or nonionic, and thehydrocarbon chain is hydrophobic. Surfactants are used in a variety ofproducts, including detergents and cleaners, and are also used asauxiliaries for textiles, leather and paper, in chemical processes, incosmetics and pharmaceuticals, in the food industry and in agriculture.In addition, they can be used to aid in the extraction and isolation ofcrude oils which are found in hard to access environments or as wateremulsions.

There are four types of surfactants characterized by varying uses.Anionic surfactants have detergent-like activity and are generally usedfor cleaning applications. Cationic surfactants contain long chainhydrocarbons and are often used to treat proteins and synthetic polymersor are components of fabric softeners and hair conditioners. Amphotericsurfactants also contain long chain hydrocarbons and are typically usedin shampoos. Non-ionic surfactants are generally used in cleaningproducts.

Vector: The term “vector” as used herein refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of vector is a “plasmid,” which refers to acircular double-stranded DNA loop into which additional DNA segments maybe ligated. Other vectors include cosmids, bacterial artificialchromosomes (BACs) and yeast artificial chromosomes (YACs). Another typeof vector is a viral vector, wherein additional DNA segments may beligated into the viral genome (discussed in more detail below). Certainvectors are capable of autonomous replication in a host cell into whichthey are introduced (e.g., vectors having an origin of replication whichfunctions in the host cell). Other vectors can be integrated into thegenome of a host cell upon introduction into the host cell, and arethereby replicated along with the host genome. Moreover, certainpreferred vectors are capable of directing the expression of genes towhich they are operatively linked. Such vectors are referred to hereinas “recombinant expression vectors” (or simply, “expression vectors”). Avector can also include one or more selectable marker genes and othergenetic elements known in the art.

Wax: A variety of fatty acid esters which form solids or pliablesubstances under an identified set of physical conditions. Fatty acidesters that are termed waxes generally have longer carbon chains thanfatty acid esters that are not waxes. For example, a wax generally formsa pliable substance at room temperature.

Fatty ester: Includes any ester made from a fatty acid. The carbonchains in fatty acids can contain any combination of the modificationsdescribed herein. For example, the carbon chain can contain one or morepoints of unsaturation, one or more points of branching, includingcyclic branching, and can be engineered to be short or long. Any alcoholcan be used to form fatty acid esters, for example alcohols derived fromthe fatty acid biosynthetic pathway, alcohols produced by the productionhost through non-fatty acid biosynthetic pathways, and alcohols that aresupplied in the fermentation broth.

Fatty acid: Includes products or derivatives thereof made in part fromthe fatty acid biosynthetic pathway of the host organism. The fatty acidbiosynthetic pathway includes fatty acid synthase enzymes which can beengineered as described herein to produce fatty acid derivatives, and insome examples can be expressed with additional enzymes to produce fattyacid derivatives having desired carbon chain characteristics. Exemplaryfatty acid derivatives include for example, short and long chainalcohols, hydrocarbons, and fatty acid esters including waxes.

General Methods for Engineering Microorganisms to Produce Carbon-BasedProducts

The methods of the invention are based on principles of metabolicengineering, and uses, e.g., engineered pathways as described in, e.g.,WO 2007/136762 and WO 2007/139925 (each of which is incorporated byreference in its entirety for all purposes) to make products from energycaptured by photoautotrophic organisms. Generally, carbon-based productsof interest are produced by expressing a gene or a set of genes asdescribed in FIG. 1 in a photoautotrophic microorganism, e.g.,cyanobacteria, as described herein. Plasmids are constructed to expressvarious proteins that are useful in production of carbon-based products,as described in the Examples herein, e.g., Example 1. The constructs canbe synthetically made or made using standard molecular biology methodsand all the cloned genes are put under the control of constitutivepromoters or inducible promoters. Plasmids containing the genes ofinterest are transformed into the host and corresponding transformantsare selected in LB plate supplemented with antibiotics such asspectinomycin, carbenicillin, etc. Using standard molecular biologytechniques, cells in which a nucleic acid molecule has been introducedare transformed to express or over-express desired genes while othernucleic acid molecules are attenuated or functionally deleted.Transformation techniques by which a nucleic acid molecule can beintroduced into such a cell, including, but not limited to, transfectionwith viral vectors, conjugation, transformation with plasmid vectors,and introduction of naked DNA by electroporation, lipofection, andparticle gun acceleration. Transformants are inoculated into a suitablemedium. The samples containing the transformants are grown at suitabletemperatures in a shaker until they reach at certain OD. The cells arethen spun down at and the cell pellets are suspended. Separationtechniques allows for the sample to be subjected to GC/MS analysis.Total yield is determined.

Selected or Engineered Microorganisms for the Production of Carbon-BasedProducts of Interest

Microorganism: Includes prokaryotic and eukaryotic microbial speciesfrom the Domains Archaea, Bacteria and Eucarya, the latter includingyeast and filamentous fungi, protozoa, algae, or higher Protista. Theterms “microbial cells” and “microbes” are used interchangeably with theterm microorganism.

A variety of host organisms can be transformed to produce a product ofinterest. Photoautotrophic organisms include eukaryotic plants andalgae, as well as prokaryotic cyanobacteria, green-sulfur bacteria,green non-sulfur bacteria, purple sulfur bacteria, and purple non-sulfurbacteria.

Suitable organisms include extremophiles that withstand variousenvironmental parameters such as temperature, radiation, pressure,gravity, vacuum, desiccation, salinity, pH, oxygen tension, andchemicals. They include hyperthermophiles, which grow at or above 80° C.such as Pyrolobus fumarii; thermophiles, which grow between 60-80° C.such as Synechococcus lividis; mesophiles, which grow between 15-60° C.and psychrophiles, which grow at or below 15° C. such as Psychrobacterand some insects. Radiation tolerant organisms include Deinococcusradiodurans. Pressure tolerant organisms include piezophiles orbarophiles which tolerate pressure of 130 MPa. Hypergravity (e.g., >1 g)hypogravity (e.g., <1 g) tolerant organisms are also contemplated.Vacuum tolerant organisms include tardigrades, insects, microbes andseeds. Dessicant tolerant and anhydrobiotic organisms include xerophilessuch as Artemia salina; nematodes, microbes, fungi and lichens. Salttolerant organisms include halophiles (e.g., 2-5 M NaCl) Halobacteriaceaand Dunaliella salina. pH tolerant organisms include alkaliphiles suchas Natronobacterium, Bacillus firmus OF4, Spirulina spp. (e.g., pH>9)and acidophiles such as Cyanidium caldarium, Ferroplasma sp. (e.g., lowpH). Anaerobes, which cannot tolerate O₂ such as Methanococcusjannaschii; microaerophils, which tolerate some O₂ such as Clostridiumand aerobes, which require O₂ are also contemplated. Gas tolerantorganisms, which tolerate pure CO₂ include Cyanidium caldarium and metaltolerant organisms include metalotolerants such as Ferroplasmaacidarmanus (e.g., Cu, As, Cd, Zn), Ralstonia sp. CH34 (e.g., Zn, Co,Cd, Hg, Pb). Gross, Michael. Life on the Edge: Amazing CreaturesThriving in Extreme Environments. New York: Plenum (1998) and Seckbach,J. “Search for Life in the Universe with Terrestrial Microbes WhichThrive Under Extreme Conditions.” In Cristiano Batalli Cosmovici, StuartBowyer, and Dan Wertheimer, eds., Astronomical and Biochemical Originsand the Search for Life in the Universe, p. 511. Milan: EditriceCompositori (1997).

Plants include but are not limited to the following genera: Arabidopsis,Beta, Glycine, Jatropha, Miscanthus, Panicum, Phalaris, Populus,Saccharum, Salix, Simmondsia and Zea.

Algae and cyanobacteria include but are not limited to the followinggenera:

Acanthoceras, Acanthococcus, Acaryochloris, Achnanthes, Achnanthidium,Actinastrum, Actinochloris, Actinocyclus, Actinotaenium, Amphichrysis,Amphidinium, Amphikrikos, Amphipleura, Amphiprora, Amphithrix, Amphora,Anabaena, Anabaenopsis, Aneumastus, Ankistrodesmus, Ankyra, Anomoeoneis,Apatococcus, Aphanizomenon, Aphanocapsa, Aphanochaete, Aphanothece,Apiocystis, Apistonema, Arthrodesmus, Artherospira, Ascochloris,Asterionella, Asterococcus, Audouinella, Aulacoseira, Bacillaria,Balbiania, Bambusina, Bangia, Basichlamys, Batrachospermum, Binuclearia,Bitrichia, Blidingia, Botrdiopsis, Botrydium, Botryococcus,Botryosphaerella, Brachiomonas, Brachysira, Brachytrichia, Brebissonia,Bulbochaete, Bumilleria, Bumilleriopsis, Caloneis, Calothrix,Campylodiscus, Capsosiphon, Carteria, Catena, Cavinula, Centritractus,Centronella, Ceratium, Chaetoceros, Chaetochloris, Chaetomorpha,Chaetonella, Chaetonema, Chaetopeltis, Chaetophora, Chaetosphaeridium,Chamaesiphon, Chara, Characiochloris, Characiopsis, Characium, Charales,Chilomonas, Chlainomonas, Chlamydoblepharis, Chlamydocapsa,Chlamydomonas, Chlamydomonopsis, Chlamydomyxa, Chlamydonephris,Chlorangiella, Chlorangiopsis, Chlorella, Chlorobotrys, Chlorobrachis,Chlorochytrium, Chlorococcum, Chlorogloea, Chlorogloeopsis,Chlorogonium, Chlorolobion, Chloromonas, Chlorophysema, Chlorophyta,Chlorosaccus, Chlorosarcina, Choricystis, Chromophyton, Chromulina,Chroococcidiopsis, Chroococcus, Chroodactylon, Chroomonas, Chroothece,Chrysamoeba, Chrysapsis, Chrysidiastrum, Chrysocapsa, Chrysocapsella,Chrysochaete, Chrysochromulina, Chrysococcus, Chrysocrinus,Chrysolepidomonas, Chrysolykos, Chrysonebula, Chrysophyta, Chrysopyxis,Chrysosaccus, Chrysophaerella, Chrysostephanosphaera, Clodophora,Clastidium, Closteriopsis, Closterium, Coccomyxa, Cocconeis,Coelastrella, Coelastrum, Coelosphaerium, Coenochloris, Coenococcus,Coenocystis, Colacium, Coleochaete, Collodictyon, Compsogonopsis,Compsopogon, Conjugatophyta, Conochaete, Coronastrum, Cosmarium,Cosmioneis, Cosmocladium, Crateriportula, Craticula, Crinalium,Crucigenia, Crucigeniella, Cryptoaulax, Cryptomonas, Cryptophyta,Ctenophora, Cyanodictyon, Cyanonephron, Cyanophora, Cyanophyta,Cyanothece, Cyanothomonas, Cyclonexis, Cyclostephanos, Cyclotella,Cylindrocapsa, Cylindrocystis, Cylindrospermum, Cylindrotheca,Cymatopleura, Cymbella, Cymbellonitzschia, Cystodinium Dactylococcopsis,Debarya, Denticula, Dermatochrysis, Dermocarpa, Dermocarpella,Desmatractum, Desmidium, Desmococcus, Desmonema, Desmosiphon,Diacanthos, Diacronema, Diadesmis, Diatoma, Diatomella, Dicellula,Dichothrix, Dichotomococcus, Dicranochaete, Dictyochloris, Dictyococcus,Dictyosphaerium, Didymocystis, Didymogenes, Didymosphenia, Dilabifilum,Dimorphococcus, Dinobryon, Dinococcus, Diplochloris, Diploneis,Diplostauron, Distrionella, Docidium, Drapamaldia, Dunaliella,Dysmorphococcus, Ecballocystis, Elakatothrix, Ellerbeckia, Encyonema,Enteromorpha, Entocladia, Entomoneis, Entophysalis, Epichrysis,Epipyxis, Epithemia, Eremosphaera, Euastropsis, Euastrum, Eucapsis,Eucocconeis, Eudorina, Euglena, Euglenophyta, Eunotia, Eustigmatophyta,Eutreptia, Fallacia, Fischerella, Fragilaria, Fragilariforma, Franceia,Frustulia, Curcilla, Geminella, Genicularia, Glaucocystis, Glaucophyta,Glenodiniopsis, Glenodinium, Gloeocapsa, Gloeochaete, Gloeochrysis,Gloeococcus, Gloeocystis, Gloeodendron, Gloeomonas, Gloeoplax,Gloeothece, Gloeotila, Gloeotrichia, Gloiodictyon, Golenkinia,Golenkiniopsis, Gomontia, Gomphocymbella, Gomphonema, Gomphosphaeria,Gonatozygon, Gongrosia, Gongrosira, Goniochloris, Gonium, Gonyostomum,Granulochloris, Granulocystopsis, Groenbladia, Gymnodinium, Gymnozyga,Gyrosigma, Haematococcus, Hafniomonas, Hallassia, Hammatoidea, Hannaea,Hantzschia, Hapalosiphon, Haplotaenium, Haptophyta, Haslea, Hemidinium,Hemitoma, Heribaudiella, Heteromastix, Heterothrix, Hibberdia,Hildenbrandia, Hillea, Holopedium, Homoeothrix, Hormanthonema,Hormotila, Hyalobrachion, Hyalocardium, Hyalodiscus, Hyalogonium,Hyalotheca, Hydrianum, Hydrococcus, Hydrocoleum, Hydrocoryne,Hydrodictyon, Hydrosera, Hydrurus, Hyella, Hymenomonas, Isthmochloron,Johannesbaptistia, Juranyiella, Karayevia, Kathablepharis, Katodinium,Kephyrion, Keratococcus, Kirchneriella, Klebsormidium, Kolbesia,Koliella, Komarekia, Korshikoviella, Kraskella, Lagerheimia, Lagynion,Lamprothamnium, Lemanea, Lepocinclis, Leptosira, Lobococcus, Lobocystis,Lobomonas, Luticola, Lyngbya, Malleochloris, Mallomonas, Mantoniella,Marssoniella, Martyana, Mastigocoleus, Gastogloia, Melosira,Merismopedia, Mesostigma, Mesotaenium, Micractinium, Micrasterias,Microchaete, Microcoleus, Microcystis, Microglena, Micromonas,Microspora, Microthamnion, Mischococcus, Monochrysis, Monodus,Monomastix, Monoraphidium, Monostroma, Mougeotia, Mougeotiopsis,Myochloris, Myromecia, Myxosarcina, Naegeliella, Nannochloris,Nautococcus, Navicula, Neglectella, Neidium, Nephroclamys, Nephrocytium,Nephrodiella, Nephroselmis, Netrium, Nitella, Nitellopsis, Nitzschia,Nodularia, Nostoc, Ochromonas, Oedogonium, Oligochaetophora, Onychonema,Oocardium, Oocystis, Opephora, Ophiocytium, Orthoseira, Oscillatoria,Oxyneis, Pachycladella, Palmella, Palmodictyon, Pnadorina, Pannus,Paralia, Pascherina, Paulschulzia, Pediastrum, Pedinella, Pedinomonas,Pedinopera, Pelagodictyon, Penium, Peranema, Peridiniopsis, Peridinium,Peronia, Petroneis, Phacotus, Phacus, Phaeaster, Phaeodermatium,Phaeophyta, Phaeosphaera, Phaeothamnion, Phormidium, Phycopeltis,Phyllariochloris, Phyllocardium, Phyllomitas, Pinnularia, Pitophora,Placoneis, Planctonema, Planktosphaeria, Planothidium, Plectonema,Pleodorina, Pleurastrum, Pleurocapsa, Pleurocladia, Pleurodiscus,Pleurosigma, Pleurosira, Pleurotaenium, Pocillomonas, Podohedra,Polyblepharides, Polychaetophora, Polyedriella, Polyedriopsis,Polygoniochloris, Polyepidomonas, Polytaenia, Polytoma, Polytomella,Porphyridium, Posteriochromonas, Prasinochloris, Prasinocladus,Prasinophyta, Prasiola, Prochlorphyta, Prochlorothrix, Protoderma,Protosiphon, Provasoliella, Prymnesium, Psammodictyon, Psammothidium,Pseudanabaena, Pseudenoclonium, Psuedocarteria, Pseudochate,Pseudocharacium, Pseudococcomyxa, Pseudodictyosphaerium,Pseudokephyrion, Pseudoncobyrsa, Pseudoquadrigula, Pseudosphaerocystis,Pseudostaurastrum, Pseudostaurosira, Pseudotetrastrum, Pteromonas,Punctastruata, Pyramichlamys, Pyramimonas, Pyrrophyta, Quadrichloris,Quadricoccus, Quadrigula, Radiococcus, Radiofilum, Raphidiopsis,Raphidocelis, Raphidonema, Raphidophyta, Peimeria, Rhabdoderma,Rhabdomonas, Rhizoclonium, Rhodomonas, Rhodophyta, Rhoicosphenia,Rhopalodia, Rivularia, Rosenvingiella, Rossithidium, Roya, Scenedesmus,Scherffelia, Schizochlamydella, Schizochlamys, Schizomeris, Schizothrix,Schroederia, Scolioneis, Scotiella, Scotiellopsis, Scourfieldia,Scytonema, Selenastrum, Selenochloris, Sellaphora, Semiorbis,Siderocelis, Diderocystopsis, Dimonsenia, Siphononema, Sirocladium,Sirogonium, Skeletonema, Sorastrum, Spermatozopsis, Sphaerellocystis,Sphaerellopsis, Sphaerodinium, Sphaeroplea, Sphaerozosma,Spiniferomonas, Spirogyra, Spirotaenia, Spirulina, Spondylomorum,Spondylosium, Sporotetras, Spumella, Staurastrum, Stauerodesmus,Stauroneis, Staurosira, Staurosirella, Stenopterobia, Stephanocostis,Stephanodiscus, Stephanoporos, Stephanosphaera, Stichococcus,Stichogloea, Stigeoclonium, Stigonema, Stipitococcus, Stokesiella,Strombomonas, Stylochrysalis, Stylodinium, Styloyxis, Stylosphaeridium,Surirella, Sykidion, Symploca, Synechococcus, Synechocystis, Synedra,Synochromonas, Synura, Tabellaria, Tabularia, Teilingia, Temnogametum,Tetmemorus, Tetrachlorella, Tetracyclus, Tetradesmus, Tetraedriella,Tetraedron, Tetraselmis, Tetraspora, Tetrastrum, Thalassiosira,Thamniochaete, Thorakochloris, Thorea, Tolypella, Tolypothrix,Trachelomonas, Trachydiscus, Trebouxia, Trentepholia, Treubaria,Tribonema, Trichodesmium, Trichodiscus, Trochiscia, Tryblionella,Ulothrix, Uroglena, Uronema, Urosolenia, Urospora, Uva, Vacuolaria,Vaucheria, Volvox, Volvulina, Westella, Woloszynskia, Xanthidium,Xanthophyta, Xenococcus, Zygnema, Zygnemopsis, and Zygonium.

Green non-sulfur bacteria include but are not limited to the followinggenera: Chloroflexus, Chloronema, Oscillochloris, Heliothrix,Herpetosiphon, Roseiflexus, and Thermomicrobium.

Green sulfur bacteria include but are not limited to the followinggenera: Chlorobium, Clathrochloris, and Prosthecochloris.

Purple sulfur bacteria include but are not limited to the followinggenera: Allochromatium, Chromatium, Halochromatium, Isochromatium,Marichromatium, Rhodovulum, Thermochromatium, Thiocapsa,Thiorhodococcus, and Thiocystis.

Purple non-sulfur bacteria include but are not limited to the followinggenera: Phaeospirillum, Rhodobaca, Rhodobacter, Rhodomicrobium,Rhodopila, Rhodopseudomonas, Rhodothalassium, Rhodospirillum,Rodovibrio, and Roseospira.

Aerobic chemolithotrophic bacteria include but are not limited tonitrifying bacteria such as Nitrobacteraceae sp., Nitrobacter sp.,Nitrospina sp., Nitrococcus sp., Nitrospira sp., Nitrosomonas sp.,Nitrosococcus sp., Nitrosospira sp., Nitrosolobus sp., Nitrosovibriosp.; colorless sulfur bacteria such as, Thiovulum sp., Thiobacillus sp.,Thiomicrospira sp., Thiosphaera sp., Thermothrix sp.; obligatelychemolithotrophic hydrogen bacteria such as Hydrogenobacter sp., ironand manganese-oxidizing and/or depositing bacteria such as Siderococcussp., and magnetotactic bacteria such as Aquaspirillum sp.

Archaeobacteria include but are not limited to methanogenicarchaeobacteria such as Methanobacterium sp., Methanobrevibacter sp.,Methanothermus sp., Methanococcus sp., Methanomicrobium sp.,Methanospirillum sp., Methanogenium sp., Methanosarcina sp.,Methanolobus sp., Methanothrix sp., Methanococcoides sp., Methanoplanussp.; extremely thermophilic Sulfur-Metabolizers such as Thermoproteussp., Pyrodictium sp., Sulfolobus sp., Acidianus sp. and othermicroorganisms such as, Bacillus subtilis, Saccharomyces cerevisiae,Streptomyces sp., Ralstonia sp., Rhodococcus sp., Corynebacteria sp.,Brevibacteria sp., Mycobacteria sp., and oleaginous yeast.

HyperPhotosynthetic conversion requires extensive genetic modification;thus, in preferred embodiments the parental photoautotrophic organismcan be transformed with exogenous DNA.

Preferred organisms for HyperPhotosynthetic conversion include:Arabidopsis thaliana, Panicum virgatum, Miscanthus giganteus, and Zeamays (plants), Botryococcus braunii, Chlamydomonas reinhardtii andDunaliela salina (algae), Synechococcus sp PCC 7002, Synechococcus sp.PCC 7942, Synechocystis sp. PCC 6803, and Thermosynechococcus elongatusBP-1 (cyanobacteria), Chlorobium tepidum (green sulfur bacteria),Chloroflexus auranticus (green non-sulfur bacteria), Chromatium tepidumand Chromatium vinosum (purple sulfur bacteria), Rhodospirillum rubrum,Rhodobacter capsulatus, and Rhodopseudomonas palusris (purple non-sulfurbacteria).

Yet other suitable organisms include synthetic cells or cells producedby synthetic genomes as described in Venter et al. US Pat. Pub. No.2007/0264688, and cell-like systems or synthetic cells as described inGlass et al. US Pat. Pub. No. 2007/0269862.

Still, other suitable organisms include microorganisms that can beengineered to fix carbon dioxide bacteria such as Escherichia coli,Acetobacter aceti, Bacillus subtilis, yeast and fungi such asClostridium ljungdahlii, Clostridium thermocellum, Penicilliumchrysogenum, Pichia pastoris, Saccharomyces cerevisiae,Schizosaccharomyces pombe, Pseudomonas fluorescens, or Zymomonasmobilis.

A common theme in selecting or engineering a suitable organism isautotrophic fixation of carbon, such as CO₂ to products. This wouldcover photosynthesis and methanogenesis. Acetogenesis, encompassing thethree types of CO₂ fixation; Calvin cycle, acetyl CoA pathway andreductive TCA pathway is also covered. The capability to use carbondioxide as the sole source of cell carbon (autotrophy) is found inalmost all major groups of prokaryotes. The CO₂ fixation pathways differbetween groups, and there is no clear distribution pattern of the fourpresently-known autotrophic pathways. Fuchs, G. 1989. Alternativepathways of autotrophic CO₂ fixation, p. 365-382. In H. G. Schlegel, andB. Bowien (ed.), Autotrophic bacteria. Springer-Verlag, Berlin, Germany.The reductive pentose phosphate cycle (Calvin-Bassham-Benson cycle)represents the CO₂ fixation pathway in almost all aerobic autotrophicbacteria, for example, the cyanobacteria.

Propagation of Selected Microoganisms

Methods for cultivation of photosynthetic organisms in liquid media andon agarose-containing plates are well known to those skilled in the art(see, e.g., websites associated with ATCC, and with the InstitutePasteur). For example, Synechococcus sp. PCC 7002 cells (available fromthe Pasteur Culture Collection of Cyanobacteria) are cultured in BG-11medium (17.65 mM NaNO3, 0.18 mM K2HPO4, 0.3 mM MgSO4, 0.25 mM CaCl2,0.03 mM citric acid, 0.03 mM ferric ammonium citrate, 0.003 mM EDTA,0.19 mM Na2CO3, 2.86 mg/L H3BO3, 1.81 mg/L MnCl2, 0.222 mg/L ZnSO4,0.390 mg/L Na2MoO4, 0.079 mg/L CuSO4, and 0.049 mg/L Co(NO3)2, pH 7.4)supplemented with 16 μg/L biotin, 20 mM MgSO4, 8 mM KCl, and 300 mM NaCl(see, e.g., website associated with the Institute Pasteur, and Price GD, Woodger F J, Badger M R, Howitt S M, Tucker L. “Identification of aSulP-type bicarbonate transporter in marine cyanobacteria. Proc Natl.Acad. Sci. USA (2004) 101(52):18228-33). Typically, cultures aremaintained at 28° C. and bubbled continuously with 5% CO2 under a lightintensity of 120 μmol photons/m2/s. Alternatively, as described inExample 1, Synechococcus sp. PCC 7002 cells are cultured in A⁺ medium aspreviously described [Frigaard N U et al. (2004) “Gene inactivation inthe cyanobacterium Synechococcus sp. PCC 7002 and the green sulfurbacterium Chlorobium tepidum using in vitro-made DNA constructs andnatural transformation,” Methods Mol. Biol., 274:325-340].

Thermosynechococcus elongatus BP-1 (available from the KazusaDNAResearch Institute, Japan) is propagated in BG11 medium supplementedwith 20 mM TES-KOH (pH 8.2) as previously described [Iwai M, Katoh H,Katayama M, Ikeuchi M. “Improved genetic transformation of thethermophilic cyanobacterium, Thermosynechococcus elongatus BP-1.” PlantCell Physiol (2004). 45(2):171-175)]. Typically, cultures are maintainedat 50° C. and bubbled continuously with 5% CO₂ under a light intensityof 38 μmol photons/m2/s. T. elongatus BP-1 can be grown in A⁺ mediumalso as described in Example 2.

Chlamydomonas reinhardtii (available from the Chlamydomonas Centerculture collection maintained by Duke University, Durham, N.C.,) aregrown in minimal salt medium consisting of 143 mg/L K2HPO4, 73 mg/LKH2PO4, 400 mg/L NH4NO3, 100 mg/L MgSO4-7H2O, 50 mg/L CaCl2-2H20, 1 mL/Ltrace elements stock, and 10 mL/L 2.0 M MOPS titrated with Tris base topH 7.6 as described (Geraghty A M, Anderson J C, Spalding M H. “A 36kilodalton limiting-CO2 induced polypeptide of Chlamydomonas is distinctfrom the 37 kilodalton periplasmic anhydrase.” Plant Physiol (1990).93:116-121). Typically, cultures are maintained at 24° C. and bubbledwith 5% CO2 in air, under a light intensity of 60 gmol photons/m2/s.

The above define typical propagation conditions. As appropriate,incubations are performed using alternate media or gas compositions,alternate temperatures (5-75° C.), and/or light fluxes (0-5500 μmolphotons/m2/s).

Light is delivered through a variety of mechanisms, including naturalillumination (sunlight), standard incandescent, fluorescent, or halogenbulbs, or via propagation in specially-designed illuminated growthchambers (for example Model LI15 Illuminated Growth Chamber (SheldonManufacturing, Inc. Cornelius, Oreg.). For experiments requiringspecific wavelengths and/or intensities, light is distributed via lightemitting diodes (LEDs), in which wavelength spectra and intensity can becarefully controlled (Philips).

Carbon dioxide is supplied via inclusion of solid media supplements(i.e., sodium bicarbonate) or as a gas via its distribution into thegrowth incubator or media. Most experiments are performed usingconcentrated carbon dioxide gas, at concentrations between 1 and 30%,which is directly bubbled into the growth media at velocities sufficientto provide mixing for the organisms. When concentrated carbon dioxidegas is utilized, the gas originates in pure form fromcommercially-available cylinders, or preferentially from concentratedsources including off-gas or flue gas from coal plants, refineries,cement production facilities, natural gas facilities, breweries, and thelike.

Transformation of Selected Microorganisms

Synechococcus sp. PCC 7002 cells are transformed according to theoptimized protocol previously described [Essich E S, Stevens Jr E,Porter R D “Chromosomal Transformation in the Cyanobacterium Agmenellumquadruplicatum”. J Bacteriol (1990). 172(4):1916-1922]. Cells are grownin Medium A (18 g/L NaCl, 5 g/L MgSO4. 7H2O, 30 mg/L Na2EDTA, 600 mg/LKCl, 370 mg/L CaCl2. 2H20, 1 g/L NaNO3, 50 mg/L KH2PO4, 1 g/L Trizmabase pH 8.2, 4 μg/L Vitamin B12, 3.89 mg/L FeCl3. 6H2O, 34.3 mg/L H3BO3,4.3 mg/L MnCl2.4H2O, 315 μg/L ZnCl2, 30 μg/L MoO3, 3 μg/L CuSO4.5H20,12.2 μg/L CoCl2. 6H20) [Stevens S E, Patterson C O P, and Myers J. “Theproduction of hydrogen peroxide by green algae: a survey.” J. Phycology(1973). 9:427-430] plus 5 g/L of NaNO3 to approximately 108 cells/mL.Nine volumes of cells are mixed with 1 volume of 1-10 μg/mL DNA in 0.15M NaCl/0.015 M Na₃ citrate and incubated at 27-30° C. for 3 hours beforeaddition of 1 volume of DNaseI to a final concentration of 10 μg/mL. Thecells are plated in 2.5 mL of 0.6% medium A overlay agar that wastempered at 45° C. and incubated. Cells are challenged with antibioticby under-laying 2.0 mL of 0.6% medium A agar containing appropriateconcentration of antibiotic with a sterile Pasteur pipette.Transformants are picked 3-4 days later. Selections are typicallyperformed using 200 μg/ml kanamycin, 8 μg/ml chloramphenicol, 10 μg/mlspectinomycin on solid media, whereas 150 μg/ml kanamycin, 7 μg/mlchloramphenicol, and 5 μg/ml spectinomycin are employed in liquid media.

T. elongatus BP-1 cells are transformed according to the optimizedprotocol previously described (Iwai M, Katoh H, Katayama M, andIkeuchi).

E. coli are transformed using standard techniques known to those skilledin the art, including heat shock of chemically competent cells andelectroporation [Berger and Kimmel, Guide to Molecular CloningTechniques, Methods in Enzymology volume 152 Academic Press, Inc., SanDiego, Calif.; Sambrook et al. (1989) Molecular Cloning—A LaboratoryManual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold SpringHarbor Press, N.Y.; and Current Protocols in Molecular Biology, F. M.Ausubel et al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (through andincluding the 1997 Supplement)].

The biosynthetic pathways as described herein are first tested andoptimized using episomal plasmids described above. Non-limitingoptimizations include promoter swapping and tuning, ribosome bindingsite manipulation, alteration of gene order (e.g., gene ABC versus BAC,CBA, CAB, BCA), co-expression of molecular chaperones, random ortargeted mutagenesis of gene sequences to increase or decrease activity,folding, or allosteric regulation, expression of gene sequences fromalternate species, codon manipulation, addition or removal ofintracellular targeting sequences such as signal sequences, and thelike.

Each gene or engineered nucleic acid is optimized individually, oralternately, in parallel. Functional promoter and gene sequences aresubsequently integrated into the E. coli chromosome to enable stablepropagation in the absence of selective pressure (i.e., inclusion ofantibiotics) using standard techniques known to those skilled in theart.

FIG. 1 lists genes involved in the production of carbon-based productsof interest, related to associated pathways, Enzyme Commission (EC)Numbers, exemplary gene names, source organism, GenBank accessionnumbers, and homologs from alternate sources. When the parental organismencodes a gene with the indicated enzymatic activity, it is useful tooverexpress these components or at least attenuate these components asindicated. In one embodiment, the native enzyme sequence isoverexpressed or attenuated. In preferred embodiments, it is useful tooverexpress or attenuate an exogenous gene, which allows for moreexplicit regulatory control in the bioprocess and a means to potentiallymitigate the effects of central metabolism regulation, which is focusedaround the native genes explicitly.

Ethanol Production

In one aspect, alcohols such as ethanol, propanol, isopropanol, butanol,fatty alcohols, other such carbon-based products of interest areproduced. FIG. 2 provides one pathway to produce ethanol, succinate andderivatives thereof.

To date, current yields of ethanol produced in cyanobacteria are notsuited for commercial production at 1.3 mM per OD₇₃₀ per day, asdisclosed in WO 2007/084477, or 1.7 μmol of ethanol per mg ofchlorophyll per hour, as shown in U.S. Pat. No. 6,699,696.

The present invention, therefore, provides methods to produce a hostcell capable of CO2 fixation that produces biofuels, e.g., ethanol at acommercial level, e.g., at between about 50 and 150 g/L in about a 48hour period. In certain embodiments, the rate of ethanol productivity isin the range of about 2.5 g/L-hr to about 5 g/L-hr. In one embodiment, ahost cell capable of CO2 fixation such as a cyanobacterium Synechococcussp. PCC 7002 is engineered to express genes such as pdc and/or adh asdisclosed. Such recombinant microorganism encodes PDC activityconverting pyruvic acid to acetoaldehyde and/or ADH activity convertingacetoaldehyde to ethanol. The transformed microorganism's ability to fixCO2 obviates the need to supplement with either sugars or biomass.Accordingly, the microorganisms of the present invention are attractivealternatives to produce biofuels. The present invention provides the w/vof ethanol to be at least 50, or at least 60 or at least 70 or at least80 or at least 90 or at least 100 or at least 125 or at least 150 g/L orotherwise produced in commercial scale.

Enzyme Selection & Optimal Enzymes

Currently, fermentative products such as ethanol, butanol, lactic acid,formate, acetate produced in biological organisms employ aNADH-dependent processes. NAD is used to break down glucose or othersugar sources to form NADH. NADH is recycled during fermentation to NAD⁺to allow further sugar breakdown, which results in fermentativebyproducts. During photosynthesis, however, the cell forms NADPH, whichis used mostly for biosynthetic operations in biological organisms,e.g., cell for growth, division, and for building up chemical storessuch as glycogen, sucrose, and other macromolecules. Fermentativeproducts are produced in the light, but in small quantities.

Using natural or engineered enzymes that utilize NADPH as a source ofreducing power instead of NADH would allow direct use of photosyntheticreducing power towards formation of normally fermentative byproducts.Accordingly, the present invention provides methods for producingfermentative products such as ethanol by expressing NADPH-dependentenzymes. This is an improvement from previous methods of using organismssuch as algae to build stores of chemicals, which are subsequently usedto make fermentation products at night or the extraneous use of separateorganisms. In effect, fermentative products are formed at higherefficiencies directly in the light during photosynthesis. In addition,the obligatory production of macromolecules at high concentration isalleviated during the day, by producing such products directly duringthe day.

NADPH-dependent enzymes that produce normally fermented products arerare in nature. Accordingly, in certain aspects of the invention,ethanol is produced in organisms expressing or modified to expressMoorella sp. HUC22-1 or a homolog thereof including at least threealcohol dehydrogenases such as AdhA (NCBI Accession YP_(—)430754). Thisenzyme has previously shown to preferentially use NADP as a cofactor asopposed to NAD and produce ethanol at high rates from acetaldehyde[“Characterization of enzymes involved in the ethanol production ofMoorella sp. HUC22-1”]. By co-expressing this gene in selectedorganisms, such as cyanobacteria, NADPH₂ formed during photosynthesiscan be used directly to form ethanol in these organisms. Alternatively,enzymes that naturally use NADH can be engineered using establishedprotein engineering techniques to require NADPH₂ rather than NADH.

In certain embodiments, NADPH-dependent AdhA from Moorella isco-expressed with pyruvate decarboxylase from Zymomonas mobilis incyanobacteria in order to derive an efficient process for ethanolproduction dependent upon NADPH as a cofactor rather than thetraditional NADH. Such transgenic organisms are able to make ethanolusing NADPH-dependent processes.

Isolated Polynucleotides

Accordingly, the present invention provides isolated nucleic acidmolecules for the adhA gene and variants thereof. The full-lengthnucleic acid sequence for this gene, encoding the enzyme NADP-dependantalcohol dehydrogenase, EC 1.1.1.2, has been identified and sequenced.SEQ ID NO:1 represents the codon- and expression-optimized codingsequence for the Moorella sp. HUC22-1 adhA gene of the presentinvention.

The present invention provides a nucleic acid molecule comprising orconsisting of a sequence which is a codon and expression optimizedversion of the wild-type adhA gene. In a further embodiment, the presentinvention provides a nucleic acid molecule and homologs, variants andderivatives of SEQ ID NO:1 comprising or consisting of a sequence whichis a variant of the adhA gene having at least 77.1% identity to SEQ IDNO: 1. The nucleic acid sequence can be preferably 78%, 79%, 80%,81%-85%, 90%-95%, 96%-98%, 99%, 99.9% or even higher identity to SEQ IDNO: 1.

In another embodiment, the invention provides a nucleic acid moleculeencoding a polypeptide having the amino acid sequence of SEQ ID NO:2.

The present invention also provides nucleic acid molecules thathybridize under stringent conditions to the above-described nucleic acidmolecules. As defined above, and as is well known in the art, stringenthybridizations are performed at about 25° C. below the thermal meltingpoint (T_(m)) for the specific DNA hybrid under a particular set ofconditions, where the T_(m) is the temperature at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Stringentwashing is performed at temperatures about 5° C. lower than the T_(m)for the specific DNA hybrid under a particular set of conditions.

Nucleic acid molecules comprising a fragment of any one of theabove-described nucleic acid sequences are also provided. Thesefragments preferably contain at least 20 contiguous nucleotides. Morepreferably the fragments of the nucleic acid sequences contain at least25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or even more contiguousnucleotides.

The nucleic acid sequence fragments of the present invention displayutility in a variety of systems and methods. For example, the fragmentsmay be used as probes in various hybridization techniques. Depending onthe method, the target nucleic acid sequences may be either DNA or RNA.The target nucleic acid sequences may be fractionated (e.g., by gelelectrophoresis) prior to the hybridization, or the hybridization may beperformed on samples in situ. One of skill in the art will appreciatethat nucleic acid probes of known sequence find utility in determiningchromosomal structure (e.g., by Southern blotting) and in measuring geneexpression (e.g., by Northern blotting). In such experiments, thesequence fragments are preferably detectably labeled, so that theirspecific hydridization to target sequences can be detected andoptionally quantified. One of skill in the art will appreciate that thenucleic acid fragments of the present invention may be used in a widevariety of blotting techniques not specifically described herein.

It should also be appreciated that the nucleic acid sequence fragmentsdisclosed herein also find utility as probes when immobilized onmicroarrays. Methods for creating microarrays by deposition and fixationof nucleic acids onto support substrates are well known in the art.Reviewed in DNA Microarrays: A Practical Approach (Practical ApproachSeries), Schena (ed.), Oxford University Press (1999) (ISBN:0199637768); Nature Genet. 21(1)(suppl):1-60 (1999); Microarray Biochip:Tools and Technology, Schena (ed.), Eaton PublishingCompany/BioTechniques Books Division (2000) (ISBN: 1881299376), thedisclosures of which are incorporated herein by reference in theirentireties. Analysis of, for example, gene expression using microarrayscomprising nucleic acid sequence fragments, such as the nucleic acidsequence fragments disclosed herein, is a well-established utility forsequence fragments in the field of cell and molecular biology. Otheruses for sequence fragments immobilized on microarrays are described inGerhold et al., Trends Biochem. Sci. 24:168-173 (1999) and Zweiger,Trends Biotechnol. 17:429-436 (1999); DNA Microarrays: A PracticalApproach (Practical Approach Series), Schena (ed.), Oxford UniversityPress (1999) (ISBN: 0199637768); Nature Genet. 21(1)(suppl):1-60 (1999);Microarray Biochip: Tools and Technology, Schena (ed.), Eaton PublishingCompany/BioTechniques Books Division (2000) (ISBN: 1881299376), thedisclosures of each of which is incorporated herein by reference in itsentirety.

In another embodiment, isolated nucleic acid molecules encoding theNADPH-dependent AdhA polypeptide comprising alcohol dehydrogenaseactivity are provided. As is well known in the art, enzyme activitiescan be measured in various ways. For example, the pyrophosphorolysis ofOMP may be followed spectroscopically. Grubmeyer et al., J. Biol. Chem.268:20299-20304 (1993). Alternatively, the activity of the enzyme can befollowed using chromatographic techniques, such as by high performanceliquid chromatography. Chung and Sloan, J. Chromatogr. 371:71-81 (1986).As another alternative the activity can be indirectly measured bydetermining the levels of product made from the enzyme activity. Theselevels can be measured with techniques including aqueouschloroform/methanol extraction as known and described in the art (cf. M.Kates (1986) Techniques of Lipidology; Isolation, analysis andidentification of Lipids. Elsevier Science Publishers, New York (ISBN:0444807322)). More modern techniques include using gas chromatographylinked to mass spectrometry (Niessen, W. M. A. (2001). Current practiceof gas chromatography—mass spectrometry. New York, N.Y: Marcel Dekker.(ISBN: 0824704738)). Additional modern techniques for identification ofrecombinant protein activity and products including liquidchromatography-mass spectrometry (LCMS), high performance liquidchromatography (HPLC), capillary electrophoresis, Matrix-Assisted LaserDesorption Ionization time of flight-mass spectrometry (MALDI-TOF MS),nuclear magnetic resonance (NMR), near-infrared (NIR) spectroscopy,viscometry (Knothe, G., R. O. Dunn, and M. O. Bagby. 1997. Biodiesel:The use of vegetable oils and their derivatives as alternative dieselfuels. Am. Chem. Soc. Symp. Series 666: 172-208), titration fordetermining free fatty acids (Komers, K., F. Skopal, and R. Stloukal.1997. Determination of the neutralization number for biodiesel fuelproduction. Fett/Lipid 99(2): 52-54), enzymatic methods (Bailer, J., andK. de Hueber. 1991. Determination of saponifiable glycerol in“bio-diesel.”Fresenius J. Anal. Chem. 340(3): 186), physicalproperty-based methods, wet chemical methods, etc. can be used toanalyze the levels and the identity of the product produced by theorganisms of the present invention. Other methods and techniques mayalso be suitable for the measurement of enzyme activity, as would beknown by one of skill in the art.

Also provided are vectors, including expression vectors, which comprisethe above nucleic acid molecules of the present invention, as describedfurther herein. In a first embodiment, the vectors include the isolatednucleic acid molecules described above. In an alternative embodiment,the vectors of the present invention include the above-described nucleicacid molecules operably linked to one or more expression controlsequences. The vectors of the instant invention may thus be used toexpress an NADPH-dependent AdhA polypeptide comprising alcoholdehydrogenase activity.

Isolated Polypeptides

According to another aspect of the present invention, isolatedpolypeptides (including muteins, allelic variants, fragments,derivatives, and analogs) encoded by the nucleic acid molecules of thepresent invention are provided. In one embodiment, the isolatedpolypeptide comprises the polypeptide sequence corresponding to SEQ IDNO:2. In an alternative embodiment of the present invention, theisolated polypeptide comprises a polypeptide sequence at least 71.1%identical to SEQ ID NO: 2. Preferably the isolated polypeptide of thepresent invention has 72%, 73%-75%, 76%-80%, 81%-90%, 95%, 96%, 97%,98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%,99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or evenhigher identity to SEQ ID NO: 2.

According to other embodiments of the present invention, isolatedpolypeptides comprising a fragment of the above-described polypeptidesequences are provided. These fragments preferably include at least 20contiguous amino acids, more preferably at least 25, 30, 35, 40, 45, 50,60, 70, 80, 90, 100 or even more contiguous amino acids.

The polypeptides of the present invention also include fusions betweenthe above-described polypeptide sequences and heterologous polypeptides.The heterologous sequences can, for example, include sequences designedto facilitate purification, e.g., histidine tags, and/or visualizationof recombinantly-expressed proteins. Other non-limiting examples ofprotein fusions include those that permit display of the encoded proteinon the surface of a phage or a cell, fusions to intrinsicallyfluorescent proteins, such as green fluorescent protein (GFP), andfusions to the IgG Fc region.

Results of Optimal Enzymes

Increased level of ethanol is observed by engineering host cells to haveNADPH-dependent alcohol dehydrogenase activity. Methods for producingincreased level of ethanol comprise expression of such NADPH-dependentadhA genes as described herein.

In certain aspects of the invention, increased levels of ethanol are atleast about 249 mg/L of ethanol is produced over 72 hours. Morepreferably, at least about 297 mg/L of ethanol is produced over 72 hours(FIG. 12).

In other aspects of the invention, methods to produce decreased levelsof acetaldehyde are disclosed. In preferred embodiments, less than about14 mg/L of acetaldehyde is produced (FIG. 13).

Still, in other aspects of the invention, methods to produce increasedamount of ethanol relative to increased OD is also disclosed. Inpreferred embodiments, at least about 36 mg/L of ethanol per OD isproduced. More preferably, at least about 47 mg/L of ethanol per OD isproduced (FIG. 14).

Accordingly, expression of such NADPH-dependent enzymes for generatingfermentative products such as ethanol is shown herein to increase levelsof ethanol, decrease levels of acetaldehyde and in effect allows forincreased ethanol production as a function of OD.

Nutrient Independence

In another aspect, in addition to CO2 and light, photoautotrophicorganisms typically require inorganic nutrient sources and vitamins.Required nutrients are generally supplemented to the growth media duringbench-scale propagation of such organisms. However, such nutrients areprohibitively expensive in the context of industrial scalebioprocessing.

Vitamin B12 is a vitamin cofactor that facilitates radical-basedreaction catalyzation. Many organisms, including Synechococcus sp. PCC7002, require external sources of Vitamin B12 for growth, which isprohibitively expensive in large-scale industrial bioprocessing. In oneembodiment, the need for Vitamin B12 is obviated by engineeringphotoautotrophic cells to express the Vitamin B12 biosynthesis pathwayas disclosed in PCT/US2008/083056, filed Nov. 10, 2008. An exemplarybiosynthesis pathway found in Salmonella typhimurium is overexpressed,including but not limited to the following genes encoding the amino acidsequences set forth in (Uroporphyrin-III C-methyltransferase (CysG), EC2.1.1.107, locus NP_(—)462380), (Sirohydrochlorin cobaltochelatase(CbiK), EC 4.99.1.3, locus NP_(—)460970), (Precorrin-2 C20methyltransferase (CbiL), EC 2.1.1.130, locus NP_(—)460969),(Precorrin3B methylase (Cb1H), EC 2.1.1.131, locus NP_(—)460972),(Bifunctional CbiG/precorrin methyltransferase (CbiG), locusNP_(—)460973), (Precorrin-4 C11-methyltransferase (CbiF), EC 2.1.1.133,locus NP_(—)460974), (Cobalamin biosynthesis protein (CbiD), locusNP_(—)460977), (NADPH-dependent precorrin-6A reductase (CbiJ), EC1.3.1.54, locus NP_(—)460971), (Precorrin-6B C5,15-methyltransferase(CbiE), EC 2.1.1.132, locus NP_(—)460976), (Precorrin-6B C12decarboxylase (CbiT), EC 2.1.1.132, locus NP_(—)460975),(Precorrin-8X-methylmutase (CbiC), EC 5.4.1.2, locus NP_(—)460978),(Cobyrinic acid A,C-diamide synthase (CbiA), EC 6.3.1.-, locusNP_(—)460980), (Cob(I)yrinic acid a,c-diamide adenosyltransferase(BtuR), EC 2.5.1.17, locus NP_(—)460677), (Cobyrinic acid synthase(CbiP), EC 6.3.5.10, locus NP_(—)460964), (Cobyric acid decarboxylase(CobD), EC 4.1.1.81, locus NP_(—)459636), (Adenosylcobinamide-phosphatesynthase (CbiB), EC 6.3.1.10, locus NP_(—)460979), (Alpha ribazole-5′-Pphosphatase (CobC), EC 3.1.3.73, locus NP_(—)459635),(Cobalamin(5′-phosphate) synthase (CobS), EC 2.7.8.26, locusNP_(—)460962), (Cobinamide phosphate guanylyl transferase (CobU), EC2.7.7.62, locus NP_(—)460963), and (Nicotinate-nucleotidedimethylbenzimidazole-P phosphoribosyl transferase (CobT), EC 2.4.2.21,locus NP_(—)460961)].

In addition, to allow for cobalt uptake and incorporation into VitaminB12, the genes encoding the cobalt transporter are overexpressed. Theexemplary cobalt transporter protein found in Salmonella typhimurium isoverexpressed and is encoded by amino acid sequences set forth in(ABC-type Co2+ transport system, permease component (CbiM), locusNP_(—)460968), (ABC-type cobalt transport system, periplasmic component(CbiN), locus NP_(—)460967), and (ABC-type cobalt transport system,permease component (CbiQ), locus NP_(—)461989).

In a preferred embodiment, photoautotrophic organisms are engineered tooverexpress Vitamin B12-independent enzymes to obviate the need for thiscofactor entirely. In most photoautotrophic organisms, only methioninesynthase (EC 2.1.1.13) and class II ribonucleotide reductases requireVitamin B12. An exemplary Vitamin B12 independent methionine synthase(EC 2.1.1.14) from Thermotoga maritime is therefore overexpressed, asset forth in PCT/US2008/083,506(5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase(MetE), locus NP_(—)229090). In addition, an exemplary class Iribonucleotide reductase (nrdAB) from Synechocystis sp. PCC 6803 isoverexpressed encoding the amino acids sequences set forth in(Ribonucleoside-diphosphate reductase, alpha subunit (NrdA), locusNP_(—)441654), (Ribonucleoside-diphosphate reductase, beta subunit(NrdB), locus NP_(—)443040).

By engineering an organism with the enzymes listed above and in FIG. 1,a photoethanolgen is produced as described more specifically in Example3. Accordingly, the present invention provides in a host cell capable ofCO₂ fixation such as a cyanobacterium Synechococcus sp. PCC 7002 that isengineered to express genes such as pdc and/or adh to produce ethanol,the host cell is engineered to be nutrient independent.

Ethanol Production Under Continuous Illumination

Normally, glycogen in microorganisms is formed in the light, to beconsumed for reducing power when it is dark. With continuousillumination, however, it may be disadvantageous to accumulatesignificant amounts of glycogen, which would draw carbon away from thedesired pathway(s), especially because there could be no dark periodlong enough to utilize the glycogen. In certain embodiments to preventglycogen synthesis during illumination, genes encoding enzyme activitiesrelated to glycogen synthesis is attenuated or eliminated entirely, tothe extent that the organism continue to be easily maintained in aviable state and be robust in fermentation conditions. Accordingly, thepresent invention provides microorganism that are at least attenuated inthe enzyme activities including, without limitation: glucose-1-phosphateadenylyltransferase (EC 2.7.7.27), glycogen synthase (EC 2.4.1.21 and EC2.4.1.11), glucose-1-phosphate uridylyltransferase (EC 2.7.7.9), and1,4-alpha-glucan branching enzyme (EC 2.4.1.18).

In certain aspects for ethanol production, the carbon that is availablefrom CO₂ fixation is directed to pyruvate as effectively as possible.Cyanobacteria can synthesize some pyruvate from carbon fixation duringillumination, using glyceraldehyde-3-phosphate derived from the Calvincycle, because they still must make biosynthetic precursors from it.However, they do so only to the extent that they require it for growth.To increase the flux to pyruvate from Calvin cycle intermediates, it isdesirable to express constitutively the genes encoding glycolyticenzymes, from the native host or from a non-native host. The choice ofgenes is made on the basis of whether allosteric regulatory effects areprojected to prevent them from exercising their full activities in theexpected metabolic context of the host. Constitutivity could be achievedby eliminating transcriptional regulation where it exists, or byexpressing the enzymes from constitutive promoters with which they arenot normally associated. Accordingly, the present invention providesethanol-producing microorganisms comprising the enzymes activitiesincluding, but without limitation: glyceraldehyde 3-phosphatedehydrogenase (EC 1.2.1.12 or EC 1.2.1.13), phosphoglycerate kinase (EC2.7.2.3), phosphoglycerate mutase (EC 5.4.2.1), enolase (EC 4.2.1.11),and pyruvate kinase (EC 2.7.1.40).

The present invention also provides additional enzyme activities for theconversion of pyruvate to ethanol. In certain embodiments, suchconversion can be carried out by at least four distinct pathways: 1) thepyruvate decarboxylase pathway, 2) the pyruvate dehydrogenase pathway,3) the pyruvate oxidase pathway, and 4) the pyruvate formate-lyasepathway. The enzyme activities required for the pyruvate decarboxylasepathway are: pyruvate decarboxylase (EC 4.1.1.1) and alcoholdehydrogenase (EC 1.1.1.1 or EC 1.1.1.2). The enzyme activities requiredfor the pyruvate dehydrogenase pathway are: acetaldehyde dehydrogenase(EC 1.2.1.10), and alcohol dehydrogenase (EC 1.1.1.1 or EC 1.1.1.2). Theenzyme activities required for the pyruvate oxidase pathway are:pyruvate oxidase (EC 1.2.2.2), acetyl-CoA synthetase (EC 6.2.1.1),acetaldehyde dehydrogenase (EC 1.2.1.10), and alcohol dehydrogenase (EC1.1.1.1 or EC 1.1.1.2). The enzyme activities required for the pyruvateformate-lyase pathway are: pyruvate formate-lyase (EC 2.3.1.54), formatehydrogen-lyase (no EC number), acetaldehyde dehydrogenase (EC 1.2.1.10),and alcohol dehydrogenase (EC 1.1.1.1 or EC 1.1.1.2). Preferably, one ormore of these pathways is expressed constitutively or under some othercontrolled regulation.

In addition to providing exogenous genes or endogenous genes with novelregulation, the optimization of ethanol production in microorganismspreferably requires the elimination or attenuation of certain hostenzyme activities. These include, but are not limited to, pyruvateoxidase (EC 1.2.2.2), D-lactate dehydrogenase (EC 1.1.1.28), acetatekinase (EC 2.7.2.1), phosphate acetyltransferase (EC 2.3.1.8), citratesynthase (EC 2.3.3.1), phosphoenolpyruvate carboxylase (EC 4.1.1.31).The extent to which these manipulations are necessary is determined bythe observed byproducts found in the bioreactor or shake-flask. Forinstance, observation of acetate would suggest deletion of pyruvateoxidase, acetate kinase, and/or phosphotransacetylase enzyme activities.In another example, observation of D-lactate would suggest deletion ofD-lactate dehydrogenase enzyme activities, whereas observation ofsuccinate, malate, fumarate, oxaloacetate, or citrate would suggestdeletion of citrate synthase and/or PEP carboxylase enzyme activities.

Ethanol Production in Light-Dark Cycle

In alternative embodiments, the present invention is adapted so that themicroorganisms are used in systems that are suitable to run with alight-dark cycle, in which several hours of constant illumination arefollowed by several hours of relative darkness. Using such a cycle, thecomplete elimination of glycogen synthesis capability may not be aviable strategy, because the cells will require some glycogen to survivethe dark period. In this case, one of two alternative strategies can beimplemented: 1) attenuation, but not elimination, of glycogen-synthesisenzymes, so that some glycogen is still made during the light phase; or2) maximization of glycogen production during the light phase.

In one embodiment, the microorganisms are attenuated but not eliminatedin glycogen-synthesis enzyme activities. Such methods are implemented bydeleting the native glycogen-synthesis gene(s) activities and replacingthem with analogs having non-native regulation and expression at a levellower than in the native host.

In another embodiment, the microorganisms maximize glycogen productionduring the light phase. Such methods are implemented by screening forstrains with the highest glycogen content among a collection ofwild-type strains or a library of mutants of a particular strain, aftersuch strains have been grown to a suitable cell concentration andsubjected subsequently to limitation of a key nutrient such as nitrogen,phosphorus, or potassium. It would be most advantageous to utilize alight-dark cycle such that glycogen is synthesized to the maximum amountpossible in terms of dry cell weight during the light cycle, thenmetabolized to ethanol to near completion as possible during the darkcycle.

During the dark cycle, glycogen is converted to pyruvate by endogenousenzymes, but, as in the case of continuous illumination, these enzymesmay be regulated in the host such that the overall conversion proceedsat a suboptimal rate. To increase the rate of this conversion, suchregulation is defeated, either by mutagenizing and screening strains forrapid glycogen utilization in the dark or by supplying the host with thenecessary enzymes at higher expression levels and/or providing exogenousgenes encoding enzymes that are less subject to allosteric regulationthan those of the host. Accordingly, the preferred enzyme activities toachieve this effect include those listed above for the conversion ofglyceraldehyde-3-phosphate into ethanol, in addition to enzymeactivities that convert glycogen to glyceraldehyde-3-phosphate: glycogenphosphorylase (EC 2.4.1.1), phosphoglucomutase (EC 5.4.2.2),glucose-6-phosphate isomerase (EC 5.3.1.9), phosphofructokinase (EC2.7.1.11), fructose-6-phosphate aldolase (EC 4.1.2.13), andtriosephosphate isomerase (EC 5.3.1.1).

In yet another embodiment, at least one cytochrome oxidase activity isattenuated or functionally deleted. Cytochromeoxidases function totransfer electrons to oxygen in the dark. Howitt et al., made deletionsof cytochrome oxidases (CtaI, CtaII and Cyd) and were able to getstrains that did not respire in the dark but grew normally in the light.(Quinol and Cytochrome Oxidases in the Cyanobacterium Synechocystis sp.PCC 6803, [Howitt et al., Biochemistry (1998) 37(51): 17944-51]. Strainsthat lacked one oxidase respired at near-wild-type rates, whereas thosethat lacked both CtaI and Cyd did not respire. The inability to respirein the dark means more fermentative products, including ethanol andperhaps also succinate as well as 1,4-butanediol. Accordingly, thepresent invention provides a carbon fixing organism, e.g., acyanobacterium engineered wherein at least one cytochrome oxidaseactivity is attenuated, which increases production of ethanol, succinateand/or 1,4-butanediol.

Production of Ethylene, Propylene, 1-Butene, 1,3-Butadiene, AcrylicAcid, Etc.

In another aspect of the invention, ethylene is produced using analcohol dehydratase (EC 4.2.1.n), which converts ethanol to ethylene.Accordingly, ethylene is produced by expressing at least one dehydrataseactivity in a microorganism. While many dehydratases exist in nature,none has been shown to convert ethanol to ethylene (or propanol topropylene, propionic acid to acrylic acid, etc.) by dehydration. Thereappears to be no purely mechanistic reason that this cannot be the case.Many examples of biological dehydratases exist, as shown in Example 55.Biological dehydrations usually take place adjacent to an activatedcarbon atom (i.e., one with a substituent group moreelectron-withdrawing than hydrogen), which helps to reduce theactivation energy of the dehydration.

In addition, thermodynamics does not appear to be a barrier. By groupcontribution methods and by consultation of experimental values in theNIST database, the reaction ethanol→ethylene+H₂O was found to have a ΔG°of between −0.5 and −1.0 kJ/mol, thus making it mildly spontaneous. Thewater concentration will be high, but ethylene is a gas and shouldspontaneously remove itself from the vicinity of the reaction. Areaction such as that catalyzed by EC 4.2.1.54 may be of particularinterest, as it generates propenoyl-CoA (CH₂═CH—CO—SCoA) fromlactoyl-CoA (CH₃—CHOH—CO—SCoA). Replacement of —CO—SCoA with —H givesethylene (CH₂═CH₂) from ethanol (CH₃—CH₂OH). Such an enzyme can be usedas a starting point for directed evolution. Genes encoding enzymes inthe 4.2.1.x group can be identified by searching databases such asGenBank, expressed in any desired host (such as Escherichia coli, forsimplicity), and that host can be assayed for the ethanol dehydrataseactivity. A high-throughput screen is especially useful for screeningmany genes and variants of genes generated by mutagenesis. The ethanoldehydratase gene, after its development to a suitable level of activity,can then be expressed in an ethanologenic organism to enable thatorganism to produce ethylene. For instance, coexpress native or evolvedethanol dehydratase gene into an organism that already produces ethanol,then test a culture by GC analysis of offgas for ethylene productionwhich is significantly higher than without the added gene. It may bedesirable to eliminate ethanol-export proteins from the productionorganism to prevent ethanol from being secreted into the medium andpreventing its conversion to ethylene.

Preferably, a customized high-throughput screen for ethylene productionby whole cells is developed, i.e., an adaptation of a colorimetric testsuch as that described by Larue and Kurz, 1973, Plant Physiol.51:1074-5. Initially, a group of unmutagenized genes identified bydatabase DNA-sequence searches is tested for activity by expression in ahost such as E. coli. Those genes enabling ethylene production fromethanol are evolved by mutagenesis (i.e., error-prone PCR, syntheticlibraries, chemical mutagenesis, etc.) and subjected to thehigh-throughput screen.

Alternatively, genes encoding ethylene-forming enzyme activities (EfE)from various sources are expressed, e.g., Pseudomonas syringae pv.Phaseolicola (D13182), P. syringae pv. Pisi (AF101061), Ralstoniasolanacearum (AL646053). Optimizing production may require furthermetabolic engineering (improving production of alpha-ketogluterate,recycling succinate as two examples). FIG. 3 depicts ethylene productionfrom the GAP pathway using an EfE.

The production host may be of the genus Synechococcus,Thermosynechococcus, Synechocystis, or other photosyntheticmicroorganism; it may also be a commonly-used industrial organism suchas Escherichia coli, Klebsiella oxytoca, or Saccharomyces cerevisiae,among others disclosed herein.

Production of Fatty Acids

In general, carbon dioxide fixing organisms can be modified to increasethe production of acyl-ACP or acyl-CoA, reduce the catabolism of fattyacid derivatives and intermediates, or to reduce feedback inhibition atspecific points in the biosynthetic pathway. In addition to modifyingthe genes described herein additional cellular resources can be divertedto over-produce fatty acids, for example the lactate, succinate and/oracetate pathways can be attenuated, and acetyl-CoA carboxylase (ACC) canbe over expressed. The modifications to the production host describedherein can be through genomic alterations, extrachromosomal expressionsystems, or combinations thereof.

Fatty Acid Biosynthetic Pathway

In one embodiment, carbon dioxide fixing organisms such as cyanobacteriacan be engineered to express certain fatty acid synthase activities(FAS), which is a group of peptides that catalyze the initiation andelongation of acyl chains (Marrakchi et al., Biochemical Society,30:1050-1055, 2002). The acyl carrier protein (ACP) and the enzymes inthe FAS pathway control the length, degree of saturation and branchingof the fatty acids produced, which can be attenuated or over-expressed.Such enzymes include accABCD, FabD, FabH, FabG, FabA, FabZ, FabI, FabK,FabL, FabM, FabB, and FabF.

For example, carbon dioxide fixing organisms engineered with the fattyacid biosynthetic pathway uses the precursors acetyl-CoA andmalonyl-CoA. Host cells engineered to overproduce these intermediatescan serve as the starting point for subsequent genetic engineering stepsto provide the specific output products such as, fatty acid esters,hydrocarbons, fatty alcohols. Several different modifications can bemade, either in combination or individually, to the host cell to obtainincreased acetyl CoA/malonyl CoA/fatty acid and fatty acid derivativeproduction. Preferably, to increase acetyl CoA production, pdh, panK,aceEF, (encoding the E1p dehydrogenase component and the E2pdihydrolipoamide acyltransferase component of the pyruvate and2-oxoglutarate dehydrogenase complexes), fabH/fabD/fabG/acpP/fabF, andin some examples additional nucleic acid encoding fatty-acyl-CoAreductases and aldehyde decarbonylases, all under the control of aconstitutive, or otherwise controllable promoter, are expressed.Exemplary Genbank accession numbers for these genes are: pdh (BAB34380,AAC73227, AAC73226), panK (also known as coaA, AAC76952), aceEF(AAC73227, AAC73226), fabH (AAC74175), fabD (AAC74176), fabG (AAC74177),acpP (AAC74178), fabF (AAC74179).

Genes to be knocked-out or attenuated includefadE, gpsA, IdhA, pflb,adhE, pta, poxB, ackA, and/or ackB by transforming the hosts withconditionally replicative or non-replicative plasmids containing null ordeletion mutations of the corresponding genes, or by substitutingpromoter or enhancer sequences. Exemplary Genbank accession numbers forthese genes are; fadE (AAC73325), gspA (AAC76632), IdhA (AAC74462), pflb(AAC73989), adhE (AAC74323), pta (AAC75357), poxB (AAC73958), ackA(AAC75356), and ackB (BAB81430).

The resulting engineered microorganisms can be grown in a desiredenvironment, for example one with limited glycerol (less than 1% w/v inthe culture medium). As such, these microorganisms will have increasedacetyl-CoA production levels. Malonyl-CoA overproduction can be effectedby engineering the microorganism as described above, with nucleicencoding accABCD (acetyl CoA carboxylase, for example accession numberAAC73296, EC 6.4.1.2). Fatty acid overproduction can be achieved byfurther including nucleic acid encoding lipase (for example Accessionsnumbers CAA89087, CAA98876).

In some cases, acetyl-CoA carboxylase (ACC) is over-expressed toincrease the intracellular concentration thereof by at least 2-fold,such as at least 5-fold, or at least 10-fold, for example relative tonative expression levels.

In addition, the plsB (for example Accession number AAC77011) D311Emutation can be used to remove limitations on the pool of acyl-CoA.

In addition, over-expression of a sfa gene (suppressor of Fab A, forexample Accession number AAN79592) can be included in the productionhost to increase production of monounsaturated fatty acids (Rock et al,J. Bacteriology 178:5382-5387, 1996).

Expression of Thioesterase

To engineer a host cell for the production of a homogeneous populationof fatty acid derivatives, one or more endogenous genes can beattenuated or functionally deleted and one or more thioesterases can beexpressed. For example, C10 fatty acids can be produced by attenuatingthioesterase C18 (for example accession numbers AAC73596 and P0ADA1),which uses C18:1-ACP and expressing thioesterase C10 (for exampleaccession number Q39513), which uses C10-ACP, thus, resulting in arelatively homogeneous population of fatty acids that have a carbonchain length of 10. In another example, C14 fatty acid derivatives canbe produced by attenuating endogenous thioesterases that produce non-C14fatty acids and expressing the thioesterase accession number Q39473(which uses C14-ACP). In yet another example, C12 fatty acid derivativescan be produced by expressing thioesterases that use C12-ACP (forexample accession number Q41635) and attenuating thioesterases thatproduce non-C12 fatty acids. Acetyl CoA, malonyl CoA, and fatty acidoverproduction can be verified using methods known in the art, forexample by using radioactive precursors, HPLC, and GC-MS subsequent tocell lysis.

Thioesterases can be expressed in the host cell as provided in Example6. As the above exemplary genes encode preferred amino acid sequences,similar genes can be selected or optimized.

Expression of Acyl-CoA Synthase

In yet another aspect, fatty acids of various lengths can be produced ina host cell engineered by expressing or overexpressing an acyl-CoAsynthase peptides (EC 2.3.1.86), which catalyzes the conversion of fattyacids to acyl-CoA. Some acyl-CoA synthase peptides, which arenon-specific, accept other substrates in addition to fatty acids.

Fatty Alcohol Forming Peptides

In yet further aspects, hosts cells are engineered to convert acyl-CoAto fatty alcohols by expressing or overexpressing a fatty alcoholforming acyl-CoA reductase (FAR, EC 1.1.1.*), or an acyl-CoA reductases(EC 1.2.1.50) (Example 18) and alcohol dehydrogenase (EC 1.1.1.1) or acombination of the foregoing to produce fatty alcohols from acyl-CoA.Hereinafter fatty alcohol forming acyl-CoA reductase (FAR, EC 1.1.1.*),acyl-CoA reductases (EC 1.2.1.50) and alcohol dehydrogenase (EC 1.1.1.1)are collectively referred to as fatty alcohol forming peptides. Somefatty alcohol forming peptides are non-specific and catalyze otherreactions as well, for example some acyl-CoA reductase peptides acceptother substrates in addition to fatty acids.

Hydrocarbon-Based Surfactants

To produce surfactants, the host cells, e.g., that demonstrate an innateability to synthesize high levels of surfactant precursors in the formof lipids and oil is modified to include a first exogenous nucleic acidsequence encoding a protein capable of converting a fatty acid to afatty aldehyde and a second exogenous nucleic acid sequence encoding aprotein capable of converting a fatty aldehyde to an alcohol (Seevarious Examples). In some examples, the first exogenous nucleic acidsequence encodes a fatty acid reductase. In other examples, the secondexogenous nucleic acid sequence encodes mammalian microsomal aldehydereductase or long-chain aldehyde dehydrogenase. In yet another example,the first and second exogenous nucleic acid sequences are from amultienzyme complex from Arthrobacter AK 19, Rhodotorula glutinins,Acinobacler sp. strain M-1, or Candida lipolytica. In one embodiment,the first and second heterologous DNA sequences are from a multienzymecomplex from Acinobacter sp, strain M-1 or Candida lipolytica.

Additional sources of heterologous nucleic acid sequences encoding fattyacid to long-chain alcohol converting proteins that can be used insurfactant production include, but are not limited to, Mortierellaalpina (ATCC 32222), Crytococcus curvatus, (also referred to asApiotricum curvatum), Alcanivorax jadensis (T9T=DSM 12718=ATCC 700854),Acinetobacter sp. HO1-N, (ATCC 14987) and Rhodococcus opacus (PD630 DSMZ44193).

In one example, the fatty acid derivative is a saturated or unsaturatedsurfactant product having a carbon atom content limited to between 6 and36 carbon atoms. In another example, the surfactant product has a carbonatom content limited to between 24 and 32 carbon atoms.

Fatty Esters of Various Lengths

In another aspect, engineered host cells produce various lengths offatty esters. For example, alcohol O-acetyltransferase peptides (EC2.3.1.84) is expressed or overexpressed. These peptides catalyze thereaction of acetyl-CoA and an alcohol to form CoA and an acetic ester.In some embodiments, the alcohol O-acetyltransferase peptides areco-expressed with selected thioesterase peptides, FAS peptides and fattyalcohol forming peptides, thus, allowing the carbon chain length,saturation and degree of branching to be controlled. In otherembodiments, the bkd operon can be co-expressed to enable branched fattyacid precursors to be produced.

Alcohol O-acetyltransferase peptides catalyze other reactions such thatthe peptides accept other substrates in addition to fatty alcohols oracetyl-CoA thiosester, for example, other alcohols and other acyl-CoAthioesters. Modification of such enzymes and the development of assaysfor characterizing the activity of a particular alcoholO-acetyltransferase peptides are within the scope of a skilled artisan.Engineered O— acetyltransferases and O-acyltransferases can be createdthat have new activities and specificities for the donor acyl group oracceptor alcohol moiety.

Alcohol acetyl transferases (AATs, EC 2.3.1.84), which are responsiblefor acyl acetate production in various plants, can be used to producemedium chain length waxes, such as octyl octanoate, decyl octanoate,decyl decanoate, and the like. Fatty esters, synthesized from mediumchain alcohol (such as C6, C8) and medium chain acyl-CoA (or fattyacids, such as C6 or C8) have a relative low melting point. For example,hexyl hexanoate has a melting point of −55° C. and octyl octanoate has amelting point of −18 to −17° C. The low melting points of thesecompounds make them good candidates for use as biofuels.

In one embodiment, a SAAT gene is co-expressed in a production hostΔfadE with fadD from E. coli and acrl (alcohol reductase from A. baylyiADP1). Octanoic acid is provided in the fermentation media. This resultsin the production of octyl octanoate. Similarly, when the wax synthasegene from A. baylyi ADP1 is expressed in the production host instead ofthe SAAT gene, octyl octanoate is produced. Medium-chain waxes whichhave low melting points, such as octyl octanoate and octyl decanoate,are good candidates for biofuel to replace triglyceride-based biodiesel.

A recombinant SAAT gene can be synthesized using DNA 2.0 (Menlo Park,Calif.) based on the published gene sequence (accession number AF193789) and modified to eliminate the Ncdl site. The synthesized SAATgene is cloned in a vector and cotransformed into a host, which carriesafadD gene from E. coli and acrl gene from A. baylyi ADP1. Thetransformants are grown in LB medium. After induction with antibioticsand the addition of 0.02% of octanoic acid, the culture is continued at25° C. from 40 hours. After that, 3 mL of acetyl acetate is added to thewhole culture and mixed several times. The acetyl acetate phase isanalyzed by GC/MS.

Fatty Esters (Biodiesels and Waxes)

Host cells are engineered to produce fatty esters (biodiesels and waxes)from acyl-CoA and alcohols. In some examples, the alcohols are providedin the fermentation media and in other examples the host cells canprovide the alcohol as described herein. Structurally, fatty acid estershave an A and a B side, the A side of the ester is used to describe thecarbon chain contributed by the alcohol, and the B side of the ester isused to describe the carbon chain contributed by the acyl-CoA. Eitherchain can be saturated or unsaturated, branched or unbranched. In someembodiments, the engineered host cells produce fatty alcohols or shortchain alcohols. In alternative embodiments, the host cell is engineeredto produce specific acyl-CoA molecules. As used herein fatty acid estersare esters derived from a fatty acyl-thioester and an alcohol, whereinthe A side and the B side of the ester can vary in length independently.Generally, the A side of the ester is at least 1, 2, 3, 4, 5, 6, 7, or 8carbons in length, while the B side of the ester is 8, 10, 12, 14, 16,18, 20, 22, 24, or 26 carbons in length. The A side and the B side canbe straight chain or branched, saturated or unsaturated.

Increased expression of one or more wax synthases (EC 2.3.1.75) leads tothe production of fatty esters, including waxes from acyl-CoA andalcohols (Example 17). As used herein, waxes are long chain fatty acidesters, wherein the A side and the B side of the ester can vary inlength independently. Generally, the A side of the ester is at least 8,10, 12, 14, 16, 18, 20, 22, 24, or 26 carbons in length. Similarly the Bside of the ester is at least 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26carbons in length. The A side and the B side can be mono-, di-,tri-unsaturated. Wax synthase peptides are capable of catalyzing theconversion of an acyl-thioester to fatty esters and some wax synthasepeptides will catalyze other reactions for example, accept short chainacyl-CoAs and short chain alcohols to produce fatty esters. Methods toidentify wax synthase activity are provided in U.S. Pat. No. 7,118,896,which is herein incorporated by reference.

In other aspects, microorganisms are modified to produce a fattyester-based biofuel by expressing nucleic acids encoding a wax estersynthase such that is expressed so as to confer upon said microorganismthe ability to synthesize a saturated, unsaturated, or branched fattyester. In some embodiments, the wax ester synthesis proteins include,but are not limited to: fatty acid elongases, acyl-CoA reductases,acyltransferases or wax synthases, fatty acyl transferases,diacylglycerol acyltransferases, acyl-coA wax alcohol acyltransferases,bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferaseselected from a multienzyme complex from Simmondsia chinensis,Acinetobacter sp. strain ADP1 (formerly Acinetobacter calcoaceticusADP1), Pseudomonas aeruginosa, Fundibacterjadensis, Arabidopsisthaliana, or Alkaligenes eutrophus. In one embodiment, the fatty acidelongases, acyl-CoA reductases or wax synthases are from a multienzymecomplex from Alkaligenes eutrophus and other organisms known in theliterature to produce wax and fatty acid esters. Additional nucleicacids encoding wax synthesis proteins useful in fatty ester productioninclude, but are not limited to, Mortierella alpina (for example ATCC32222), Crytococcus curvatus, (also referred to as Apiotricum curvatum),Alcanivorax jadensis (for example T9T=DSM 12718=ATCC 700854),Acinetobacter sp. HO1-N, (for example ATCC 14987) and Rhodococcus opacus(for example PD630, DSMZ 44193).

Fatty esters of various length are produced, for example, the fattyester product is a saturated or unsaturated fatty ester product having acarbon atom content between 24 and 46 carbon atoms; 24 and 32 carbonatoms; or 14 and 20 carbons. In another embodiment the fatty ester isthe methyl ester of C18:1; ethyl ester of C16:1; methyl ester of C16:1;or octadecyl ester of octanol.

In another embodiment, the wax ester synthase from Acinetobacter sp.ADP1 at locus AAO17391 (described in Kalscheuer and Steinbuchel, J.Biol. Chem. 278:8075-8082, 2003, herein incorporated by reference) isused. In another example the wax ester synthase from Simmondsiachinensis, at locus AAD38041 is used.

Optionally a wax ester exporter such as a member of the FATP family isused to facilitate the release of waxes or esters into the extracellularenvironment. One example of a wax ester exporter that can be used isfatty acid (long chain) transport protein CG7400-PA, isoform A from D.melanogaster, at locus NP_(—)524723.

As described herein, the B side is contributed by a fatty acid producedfrom de novo synthesis in the host organism. In some instances where thehost is additionally engineered to make alcohols, including fattyalcohols, the A side is also produced by the host organism. In yet otherexamples the A side can be provided in the medium. As described herein,by selecting the desired thioesterase genes the B side, and when fattyalcohols are being made the A side, can be designed to be have certaincarbon chain characteristics. These characteristics include points ofunsaturation, branching, and desired carbon chain lengths. Exemplarymethods of making long chain fatty acid esters, wherein the A and B sideare produced by the production host are provided. When both the A and Bside are contributed by the production host and they are produced usingfatty acid biosynthetic pathway intermediates they will have similarcarbon chain characteristics. For example, at least 50%, 60%, 70%, or80% of the fatty acid esters produced will have A sides and B sides thatvary by 6, 4, or 2 carbons in length. The A side and the B side willalso display similar branching and saturation levels.

In one embodiment, wax esters are produced by engineering Synechococcussp. PCC 7002 to express a fatty alcohol forming acyl-CoA reductase,thioesterase, and a wax synthase. Thus, the production host producesboth the A and the B side of the ester and the structure of both sidesis influenced by the expression of the thioesterase gene A. baylyi ADP1(termed WSadpl, accessions AA017391, EC: 2.3.175). The host istransformed and selected in LB plates supplemented with antibiotics suchas kanamycin, carbenicillin or spectinomycin. Transformants areinoculated in LB and cultured in a shaker at a suitable temperature.When the cultures reach a preferred OD, an aliquot is transferred intoflasks. The culture is then placed into conical tubes and the cells arespun down. The cell pellet is then mixed with ethyl acetate. The ethylacetate extract is analyzed with GC/MS.

In addition to producing fatty alcohols for contribution to the A side,the host can produce other short chain alcohols such as ethanol,propanol, isopropanol, isobutanol, and butanol for incorporation on theA side using techniques well known in the art. For example, butanol canbe made by the host organism. To create butanol-producing cells, hostcells can be further engineered to express atoB (acetyl-CoAacetyltransferase) from E. coli K₁₂, β-hydroxybutyryl-CoA dehydrogenasefrom Butyrivibrio fibrisolvens, crotonase from Clostridium beijerinckii,butyryl CoA dehydrogenase from Clostridium beijerinckii, CoA-acylatingaldehyde dehydrogenase (ALDH) from Cladosporium fulvum, and adhEencoding an aldehyde-alcohol dehydrogenase of Clostridium acetobutylicumin an expression vector. Similarly, ethanol can be produced in aproduction host using the methods taught by Kalscheuer et al.,Microbiology 152:2529-2536, 2006, which is herein incorporated byreference.

The centane number (CN), viscosity, melting point, and heat ofcombustion for various fatty acid esters have been characterized in forexample, Knothe, Fuel Processing Technology 86:1059-1070, 2005, which isherein incorporated by reference. Using the teachings provided herein ahost cell can be engineered to produce any one of the fatty acid estersdescribed in the Knothe, Fuel Processing Technology 86:1059-1070, 2005.

Acyl-ACP, Acyl-CoA to Hydrocarbon

Various microorganisms are known to produce hydrocarbons, such asalkanes, olefins, and isoprenoids. Many of these hydrocarbons arederived from fatty acid biosynthesis. The production of thesehydrocarbons can be controlled by controlling the genes associated withfatty acid biosynthesis in the native hosts of some microorganisms. Forexample, hydrocarbon biosynthesis in the algae Botryococcus brauniioccurs through the decarbonylation of fatty aldehydes. The fattyaldehydes are produced by the reduction of fatty acyl—thioesters byfatty acyl-CoA reductase. Thus, the structure of the final alkanes canbe controlled by engineering B. braunii to express specific genes, suchas thioesterases, which control the chain length of the fatty acidsbeing channeled into alkane biosynthesis. Expressing the enzymes thatresult in branched chain fatty acid biosynthesis in B. braunii willresult in the production of branched chain alkanes. Introduction ofgenes affecting the production of desaturation of fatty acids willresult in the production of olefins. Further combinations of these genescan provide further control over the final structure of the hydrocarbonsproduced. To produce higher levels of the native or engineeredhydrocarbons, the genes involved in the biosynthesis of fatty acids andtheir precursors or the degradation to other products can be expressed,overexpressed, or attenuated. Each of these approaches can be applied tothe production of alkanes in engineered microorganisms such as Vibriofurnissi M1 and its functional homologues, which produce alkanes throughthe reduction of fatty alcohols. Each of these approaches can also beapplied to the production of the olefins produced by many strains ofMicrococcus leuteus, Stenotrophomonas maltophilia, Jeogalicoccus sp.(ATCC8456), and related microorganisms. These microorganisms producelong chain internal olefins that are derived from the head to headcondensation of fatty acid precursors. Controlling the structure andlevel of the fatty acid precursors using the methods described hereinwill result in formation of olefins of different chain length,branching, and level of saturation.

Examples 9, 10, 11 and 19 provide several alternatives in engineering amicroorganism to produce hydrocarbons such as alkane and octane.

Hydrocarbons can also be produced using evolved oxido/reductases for thereduction of primary alcohols. Primary fatty alcohols are known to beused to produce alkanes in microorganisms such as Vibrio furnissii M1(Myong-Ok, J. Bacterial., 187: 1426-1429, 2005). An NAD(P)H dependentoxido/reductase is the responsible catalyst. Synthetic NAD(P)H dependentoxidoreductases can be produced through the use of evolutionaryengineering and be expressed in production hosts to produce fatty acidderivatives. One of ordinary skill in the art will appreciate that theprocess of “evolving” a fatty alcohol reductase to have the desiredactivity is well known (Kolkman and Stemmer Nat Biotechnol 19:423-8,2001, Ness et al., Adv Protein Chem. 55:261-92, 2000, Minshull andStemmer Curr Opin Chem. Biol. 3:284-90, 1999, Huisman and Gray Curr OpinBiotechnol August; 13:352-8, 2002, and see U.S. patent publication2006/0195947). A library of NAD(P)H dependent oxidoreductases isgenerated by standard methods, such as error-prone PCR, site-specificrandom mutagenesis, site specific saturation mutagenesis, or sitedirected specific mutagenesis. Additionally, a library can be createdthrough the “shuffling” of naturally occurring NAD(P)H dependentoxidoreductase encoding sequences. The library is expressed in asuitable host, such as E. coli. Individual colonies expressing adifferent member of the oxido/reductase library is then analyzed for itsexpression of an oxido/reductase that can catalyze the reduction of afatty alcohol. For example, each cell can be assayed as a whole cellbioconversion, a cell extract, a permeabilized cell, or a purifiedenzyme. Fatty alcohol reductases are identified by the monitoring thefatty alcohol dependent oxidation of NAD(P)H spectrophotometrically orfluorometrically. Production of alkanes is monitored by GC/MS, TLC, orother methods. An oxido/reductase identified in this manner is used toproduce alkanes, alkenes, and related branched hydrocarbons. This isachieved either in vitro or in vivo. The latter is achieved byexpressing the evolved fatty alcohol reductase gene in an organism thatproduces fatty alcohols, such as those described herein. The fattyalcohols act as substrates for the alcohol reductase which would producealkanes. Other oxidoreductases can be also engineered to catalyze thisreaction, such as those that use molecular hydrogen, glutathione, FADH,or other reductive coenzymes.

Increased Fatty Acid Production

Introduction of heterologous nucleic acid sequences involved in abiosynthetic pathway for the production of hydrocarbons can be donestably or transiently into various host cells using techniques wellknown in the art, for example, electroporation, calcium phosphateprecipitation, DEAE-dextran-mediated transfection, liposome-mediatedtransfection, conjugation and transduction. For stable transformation, aDNA sequence can further include a selectable marker, such as,antibiotic resistance, for example resistance to neomycin, tetracycline,chloramphenicol, kanamycin and genes that complement auxotrophicdeficiencies.

Suitable expression control sequences for use in prokaryotic host cellsinclude, but are not limited to, promoters capable of recognizing theT4, T3, Sp6 and T7 polymerases, the PR and P_(L) promoters ofbacteriophage lambda, the trp, recA, heat shock, and lacZ promoters ofE. coli, the alpha-amylase and the sigma-specific promoters of B.subtilis, the promoters of the bacteriophages of Bacillus, Streptomycespromoters, the int promoter of bacteriophage lambda, the bla promoter ofthe beta-lactamase gene of pBR322, and the CAT promoter of thechloramphenicol acetyl transferase gene. Prokaryotic promoters arereviewed by Glick, J. Ind. Microbiol. 1:277, 1987; Watson et al,MOLECULAR BIOLOGY OF THE GENES 4th Ed., Benjamin Cummins (1987); andSambrook et al., supra.

Non-limiting examples of suitable eukaryotic promoters for use within aneukaryotic host are viral in origin and include the promoter of themouse metallothionein I gene (Hamer et al., J. Mol. Appl. Gen. 1:273,1982); the TK promoter of Herpes virus (McKnight, Cell 31:355, 1982);the SV40 early promoter (Benoist et al, Nature (London) 290:304, 1981);the Rous sarcoma virus promoter; the cytomegalovirus promoter (Foeckinget al, Gene 45:101, 1980); the yeast gal4 gene promoter (Johnston, etal, PNAS (USA) 79:6971, 1982; Silver, et al., PNAS (USA) 81:5951, 1984);and the IgG promoter (Orlandi et al, PNAS (USA) 86:3833, 1989).

In some examples a genetically modified host cell is geneticallymodified with a heterologous DNA sequence encoding a biosyntheticpathway gene product that is operably linked to a constitutive promoter.Suitable constitutive promoters are known in the art and include,constitutive adenovirus major late promoter, a constitutive MPSVpromoter, and a constitutive CMV promoter. Suitable constitutivepromoters applicable for Synechococcus sp. PCC 7002 include for example,PtacI, P-EM7, Paph2 and PaadA

The microbial host cell can be genetically modified with a heterologousnucleic acid sequence encoding a biosynthetic pathway gene product thatis operably linked to an inducible promoter. Inducible promoters arewell known in the art. Suitable inducible promoters include, but are notlimited to promoters that are affected by proteins, metabolites, orchemicals. These include: a bovine leukemia virus promoter, ametallothionein promoter, a dexamethasone-inducible MMTV promoter, aSV40 promoter, a MRP polIII promoter, a tetracycline-inducible CMVpromoter (such as the human immediate-early CMV promoter) as well asthose from the trp and lac operons.

When a host cell is genetically modified with heterologous nucleic acidsequences encoding two or more proteins involved in a biosynthesispathway to produce carbon-based products of interest, the nucleic acidsequences can be driven by a single promoter on a single vector or atleast one promoter on separate expression vectors.

In some embodiments, the intracellular concentration (e.g., theconcentration of the intermediate in the genetically modified host cell)of the biosynthetic pathway intermediate can be increased to furtherboost the yield of the final product. For example, by increasing theintracellular amount of a substrate (e.g., a primary substrate) for anenzyme that is active in the biosynthetic pathway, and the like.

In some examples the fatty acid or intermediate is produced in thecytoplasm of the cell. The cytoplasmic concentration can be increased ina number of ways, including, but not limited to, binding of the fattyacid to coenzyme A to form an acyl-CoA thioester. Additionally, theconcentration of acyl-CoAs can be increased by increasing thebiosynthesis of CoA in the cell, such as by over-expressing genesassociated with pantothenate biosynthesis (panD) or knocking out thegenes associated with glutathione biosynthesis (glutathione synthase).

Carbon Chain Modifications

FIG. 1 provides a description of the various genes that can be modulatedto alter the structure of the fatty acid derivative product and theencoded enzymes that can be used alone or in combination to make variousfatty acids and hydrocarbons. The products can be produced such thatthey contain branch points, levels of saturation, and carbon chainlength, thus, making these products desirable starting materials for usein many applications. Provided are various carbon-based products ofinterest produced by the microorganisms.

FIG. 1 also lists enzymes that are directly involved in the synthesis ofcarbon-based products, including waxes, fatty acid esters and/or fattyalcohols. To increase the production of waxes/fatty acid esters, andfatty alcohols, one or more of the enzymes can be over expressed ormutated to reduce feedback inhibition. Additionally, enzymes thatmetabolize the intermediates to make nonfatty-acid based products (sidereactions) can be functionally deleted or attenuated to increase theflux of carbon through the fatty acid biosynthetic pathway. The Examplesprovided herein describe how to engineer enzymes in the respectivepathways of host organisms to yield engineered organisms that producecarbon-based products of interest.

In other examples, the expression of exongenous FAS genes originatingfrom different species or engineered variants can be introduced into thehost cell to result in the biosynthesis of fatty acid metabolitesstructurally different (in length, branching, degree of unsaturation,etc.) as that of the native host. These heterologous gene products canbe also chosen or engineered so that they are unaffected by the naturalcomplex regulatory mechanisms in the host cell and, therefore, functionin a manner that is more controllable for the production of the desiredcommercial product. For example the FAS enzymes from Bacillus subtilis,Saccharomyces cerevisiae, Streptomyces spp, Ralstonia, Rhodococcus,Corynebacteria, Brevibacteria, Mycobacteria, oleaginous yeast, and thelike can be expressed in the production host.

A skilled artisan will appreciate that when a production host isengineered to produce a fatty acid from the fatty acid biosyntheticpathway that contains a specific level of unsaturation, branching, orcarbon chain length, the resulting engineered fatty acid can be used inthe production of fatty acid derivatives. Hence, fatty acid derivativesgenerated from the production host can display the characteristics ofthe engineered fatty acid. For example, a production host can beengineered to make branched, short chain fatty acids. Then, using theteachings provided herein relating to fatty alcohol production (i.e.,including alcohol-forming enzymes such as FAR), the production hostproduces branched, short chain fatty alcohols. Similarly, a hydrocarboncan be produced by engineering a production host to produce a fatty acidhaving a defined level of branching, unsaturation, and/or carbon chainlength, thus, producing a homogenous hydrocarbon population. Moreover,when an unsaturated alcohol, fatty acid ester or hydrocarbon is desired,the fatty acid biosynthetic pathway can be engineered to produce lowlevels of saturated fatty acids and an additional desaturase can beexpressed to lessen the saturated product production.

Saturation

In one aspect, hosts are engineered to produce unsaturated fatty acidsby over-expressing fabB, or by growing the host at low temperatures (forexample less than 37° C.). FabB has preference to cis-δ³decenoyl-ACP andresults in unsaturated fatty acid production in E. coli. Over-expressionof FabB results in the production of a significant percentage ofunsaturated fatty acids (de Mendoza et al, J. Biol. Chem., 258:2098-101,1983). These unsaturated fatty acids can then be used as intermediatesin hosts that are engineered to produce fatty acids, such as fattyalcohols, esters, waxes, olefins, alkanes, and the like. One of ordinaryskill in the art will appreciate that by attenuating fabA, orover-expressing fabB and expressing specific thioesterases (describedbelow), unsaturated fatty acid derivatives having a desired carbon chainlength can be produced. Alternatively, the repressor of fatty acidbiosynthesis, FabR (Genbank accession NP_(—)418398), can be deleted,which will also result in increased unsaturated fatty acid production inE. coli (Zhang et al., J. Biol. Chem. 277:pp. 15558, 2002.). Furtherincrease in unsaturated fatty acids is achieved by over-expression ofFabM (trans-2, cis-3-decenoyl-ACP isomerase, Genbank accession DAA05501)and controlled expression of FabK (trans-2-enoyl-ACP reductase II,Genbank accession NP_(—)357969) from Streptococcus pneumoniae (Marrakchiet al., J. Biol. Chem. 277: 44809, 2002), while deleting E. coli Fab I(trans-2-enoyl-ACP reductase, Genbank accession NP_(—)415804).Additionally, to increase the percentage of unsaturated fatty acidesters, the microorganism can also overexpressfabB (encodingβ-ketoacyl-ACP synthase I, Accessions: BAA16180, EC:2.3.1.41), Sfa(encoding a suppressor of fabA, Accession: AAC44390) and gnsA and gnsB(both encoding secG null mutant suppressors, a.k.a. cold shock proteins,Accession: ABD18647.1, AAC74076.1) over-expressed. In some examples, theendogenous fabF gene can be attenuated, thus, increasing the percentageof palmitoleate (C16:1) produced.

Fatty acids can be produced that contain branch points, cyclic moieties,and combinations thereof, using the teachings provided herein (Example11).

By inserting and expressing one or more exogenous nucleic acidsequences, microorganisms that naturally produce straight fatty acids(sFAs) can be engineered to be capable of fixing carbon dioxide andproducing branched chain fatty acids (brFAs). For example, a host suchas E. coli naturally produces straight fatty acids (sFAs). The host canalso engineered to capture light as described in, e.g.,PCT/US2008/075899, filed Sep. 10, 2008, or in PCT/US2008/083056, filedNov. 10, 2008, and several genes can be introduced and expressed thatprovide branched precursors (bkd operon) and allow initiation of fattyacid biosynthesis from branched precursors (fabH). Additionally, theorganism can express genes for the elongation of brFAs (e.g. ACP, FabF).Additionally, or alternatively, the corresponding E. coli genes thatnormally lead to sFAs and would compete with the introduced genes (e.g.FabH, FabF) can be deleted.

The branched acyl-CoAs 2-methyl-buturyl-CoA, isovaleryl-CoA andisobuturyl-CoA are the precursors of brFA. In most brFA-containingmicroorganisms they are synthesized in two steps (described in detailbelow) from branched amino acids (isoleucine, leucine and valine)[Kadena, Microbiol. Rev. 55: pp. 288, (1991)]. A microorganism can beengineered to produce brFAs, or to overproduce brFAs, by recombinantlyexpressing or over-expressing one or more of the enzymes in these twosteps. In some instances the production host may have an endogenousenzyme that can accomplish one step, in which case only enzymes involvedin the second step need to be recombinantly expressed.

The first step in forming branched fatty acids is the production of thecorresponding α-keto acids by a branched-chain amino acidaminotransferase. E. coli has such an enzyme, IvE (EC 2.6.1.42; Genbankaccession YP_(—)026247). In some examples, a heterologous branched-chainamino acid aminotransferase may not be expressed. However, E. coli IlvEor any other branched-chain amino acid aminotransferase, e.g., IlvE fromLactococcus lactis (Genbank accession AAF34406), ilvE from Pseudomonasputida (Genbank accession NP_(—)745648), or ilvE from Streptomycescoelicolor (Genbank accession NP_(—)629657) can be over-expressed in ahost microorganism, should the host's aminotransferase reaction turn outto be rate limiting.

The second step, the oxidative decarboxylation of the α-ketoacids to thecorresponding branched-chain acyl-CoA, is catalyzed by branched-chainα-keto acid dehydrogenase complexes (bkd; EC 1.2.4.4.) [Denoya et al. J.Bacteriol 177:pp. 3504, (1995)], which consist of E1α/β (decarboxylase),E2 (dihydrolipoyl transacylase) and E3 (dihydrolipoyl dehydrogenase)subunits. These subunits are similar to pyruvate and α-ketoglutaratedehydrogenase complexes. FIG. 1 lists potential bkd genes from severalmicroorganisms that can be expressed in a production host to providebranched-chain acyl-CoA precursors. Basically, every microorganism thatpossesses brFAs and/or grows on branched-chain amino acids can be usedas a source to isolate bkd genes for expression in production hosts suchas E. coli. Furthermore, E. coli naturally has the E3 component (as partof its pyruvate dehydrogenase complex; lpd, EC 1.8.1.4, Genbankaccession NP_(—)414658). Therefore, in E. coli, only the E1α/β and E2bkd genes need be expressed.

In another example, isobuturyl-CoA can be made in a production host, forexample in E. coli through the coexpression of a crotonyl-CoA reductase(Ccr, EC 1.1.1.9) and isobuturyl-CoA mutase (large subunit IcmA, EC5.4.99.2; small subunit IcmB, EC 5.4.99.13) (Han and Reynolds J.Bacteriol 179:pp. 5157, 1997). Crotonyl-CoA is an intermediate in fattyacid biosynthesis in E. coli and other microorganisms.

In addition to expression of the bkd genes, the initiation of brFAbiosynthesis utilizes β-ketoacyl-acyl-carrier-protein synthase 111(FabH, EC 2.3.1.41) with specificity for branched chain acyl CoAs (Li etal. J. Bacterial. 187:pp. 3795, 2005). FabH genes that are involved infatty acid biosynthesis of any brFA-containing microorganism can beexpressed in a production host. The Bkd and FabH enzymes from productionhosts that do not naturally make brFA may not support brFA production.Bkd and FabH, therefore, may be expressed recombinantly in these hosts.Similarly, if the endogenous level of Bkd and FabH production are notsufficient to produce brFA, these genes can be over-expressed.Additionally, other components of fatty acid biosynthesis machinery canbe expressed, including acyl carrier proteins (ACPs) and genes such asβ-ketoacyl-acyl-carrier-protein synthase II (fabF, EC 2.3.1.41). Inaddition to expressing these genes, some genes in the endogenous fattyacid biosynthesis pathway may be attenuated in the production host. Forexample, in E. coli the most likely candidates to interfere with brFAbiosynthesis are fabH (Genbank accession #NP_(—)415609) and/or fabFgenes (Genbank accession #NP_(—)415613).

As mentioned above, through the combination of expressing genes thatsupport brFA synthesis and alcohol synthesis, branched chain alcoholscan be produced. For example, when an alcohol reductase such as Acrlfrom Acinetobacter baylyi ADP1 is coexpressed with a bkd operon, E. colican synthesize isopentanol, isobutanol or 2-methyl butanol. Similarly,when Acrl is coexpressed with ccr/icm genes, E. coli can synthesizeisobutanol.

In order to convert a production host such as E. coli into an organismcapable of synthesizing ω-cyclic fatty acids (cyFAs), several genes needto be introduced and expressed that provide the cyclic precursorcyclohexylcarbonyl-CoA (Cropp et al Nature Biotech. 18:pp. 980, 2000).The genes (fabH, ACP and fabF) can then be expressed to allow initiationand elongation of ω-cyclic fatty acids (Example 13). Alternatively, thehomologous genes can be isolated from microorganisms that make cyFAs andexpressed in E. coli.

Expression of the following genes are sufficient to providecyclohexylcarbonyl-CoA in E. coli: ansJ, ansK, ansL, chcA and ansM fromthe ansatrienin gene cluster of Streptomyces collinus [Chen et al., Eur.J. Biochem. 261 (1999)] or plmJ, plmK, plmL, chcA and plmM from thephoslactomycin B gene cluster of Streptomyces sp. HK803 [Palaniappan etal., J. Biol. Chem. 278:35552 (2003)] together with the chcB gene[Patton et al. Biochem., 39:7595 (2000)] from S. collinus, S.avermitilis or S. coelicolor.

The genes (fabH, ACP andfabF) are sufficient to allow initiation andelongation of ω-cyclic fatty acids, because they can have broadsubstrate specificity. In the event that coexpression of any of thesegenes with the ansJKLM/chcAB or pmlJKLM/chcAB genes does not yieldcyFAs, fabH, ACP and/or fabF homologs from microorganisms that makecyFAs can be isolated (e.g., by using degenerate PCR primers orheterologous DNA probes) and coexpressed.

Production of Sugars

Industrial production of chemical products from biological organisms isusually accomplished by anaerobic fermentation of sugars such asglucose. Notably, photosynthetic organisms often produce polymers suchas glycogen and cellulose made up of glucose monomers in the course ofphotosynthesis. To produce fermentation products during periods ofphotosynthesis is challenging as it generally requires the cell todirect flux both towards gluconeogenesis and glycolysis simultaneously.On the other hand, using a day/night cycle to allow carbon buildup andthen fermentation of the carbon stores at night is limited by themaximum carbon storage capacities of the cell.

There are at least two major avenues to export sugars into the medium.In one embodiment, sugars and sugar phosphates such as glucose orfructose phosphates or triose phosphates are exported. Triose phosphatesinclude 3-phosphoglyceraldehyde (3PGAL) and dihydroxyacetone-phosphate(DHAP). In this case, a specific transporter is needed, which usuallyact as an anti-porter with inorganic phosphates.

In another embodiment, in order to export unphosphorylated sugars, suchas glucose, in appreciable quantities the cell is engineered todephosphorylate glucose-phosphate (e.g., phosphatase) in the cell.Diffusion through a transporter allows glucose to exit the cell.

Preferably, to prevent buildup of other storage polymers, these proteinsare expressed in cells that are attenuated in their ability to buildother storage polymers such as glycogen, starch, sucrose, cellulose, andcyanophycin.

In one aspect of the present invention, to transport 3PGAL directly outof the cells, genes are expressed that encode enzymes to facilitatetransport of the photosynthetic products from within the host cell tothe culture media. For instance, to export triose-phosphates out of thecell with concomitant import of inorganic phosphate, the followingprotein is expressed: A. thaliana triose-phosphate transporter APE2(AT5G46110.4) in a host cell of interest, e.g., cyanobacteria.

In another aspect, an antiporter or a transporter is used to transportglucose-6-phosphate or fructose-6-phosphate outside of the cell. Forexample, to export glucose-6-phosphate out of the cell with concomitantimport of inorganic phosphate, one or more of the following proteins areexpressed in a photosynthetic organism: (1) E. coli sugar phosphatetransporter UhpT (NP_(—)418122.1), (2) A. thaliana glucose-6-phosphatetransporter GPT1 (AT5G54800.1 or (3) A. thaliana glucose-6-phosphatetransporter GPT2 (AT1G61800.1).

In other embodiments, to facilitate conversion of D-fructose 6-phosphateto D-glucose-6-phosphate, a fructose-6-phosphate isomerase is introducedin the cell. For example, a glucose-6-phosphatase (e.g., GenBankAccession Nos. AAA16222, AAD 19898, 043826) is introduced into the cellto convert D-glucose 6-phosphate and H₂O to D-glucose and phosphate.

Additionally, a phosphatase enzyme activity introduced into the hostcell to dephosphorylate glucose-6-phosphate and/or glucose-1-phosphatewithin in a photosynthetic organism. In one embodiment, one or more ofthe following proteins are expressed: (1) H. sapiensglucose-6-phosphatase G6PC(P35575), (2) E. coli glucose-1-phosphataseAgp (P19926), (3) E. cloacae glucose-1-phosphatase AgpE (Q6EV19) and (4)E. coli acid phosphatase YihX (P0A8Y3).

To facilitate the diffusive efflux of the glucose made intracellularlyvia the action of the aforementioned phosphatase(s), one or more of thefollowing permeases are expressed: (1) H. sapiens glucose transporterGLUT-1, -3, or -7 (P11166, P11169, Q6PXP3), (2) S. cerevisiae hexosetransporter HXT-1, -4, or -6 (P32465, P32467, P39003), or (3) Z. mobilisglucose uniporter Glf (P21906). In certain embodiments, the followingtransporters are expressed: 2.A.1.1.32 Glucose/fructose:H+ symporter,GlcP [Zhang et al. (1989)] bacteria GlcP of Synechocystis sp. (P15729);2.A.1.1.35, the major glucose (or 2-deoxyglucose) uptake transporter,GlcP [van Wezel et al., (2005)]; and/or Q7BEC, 2.A.1.1.24 Hexose(Glucose and Fructose) transporter, PfHT1 of Plasmodium falciparum sp.097467. In other embodiments, a glucose transporter, such as the Glut-1transporter (e.g., GenBank Accession No. S77924) is introduced in thecell in order to facilitate active secretion of glucose form within thecell to the culture media. A diffusive mechanism may involve the nativebacterial transporters such as the native glucose transporters whichnormally let glucose in and/or the mammalian transporters such as Glut-1Glut-2 for diffusive efflux of glucose. Accordingly, glucose produced asa result of photosynthesis is diffused from within the cells to theculture media.

The genes corresponding to the above proteins are synthetically made andplaced downstream of constitutive and/or inducible promoters forexpression in the photosynthetic organism. The constructs can then beused to transform the organisms via linkage to a positively selectablemarker such as an antibiotic resistance gene. In certain embodiments,the above genes are integrated into the chromosome of the host cell,e.g., cyanobacteria. Preferred integration sites include genomes thatare involved in cellulose, glycogen or sucrose synthesis.

Alteration of Cellulose, Glycogen or Sucrose Synthesis

In another aspect of the invention, cells are modified to attenuate,disrupt or delete cellulose, glycogen, sucrose synthesis or acombination thereof as shown in Table 1.

TABLE 1 Synnechococcus sp. PCC 7002 Enzyme Reaction Locus celluloseUDP-glucose + A2118 synthase (1,4-beta-D-glucosyl)n = UDP +(UDP-forming) (1,4-beta-D-glucosyl)n + 1 (EC 2.4.1.12) glycogenADP-glucose + A1532 synthase (1,4-alpha-D-glucosyl)n = ADP + A2125 e.g.,glgA1, (1,4-alpha-D-glucosyl)n + 1 glgA2 (EC 2.4.1.21) sucroseUDP-glucose + A0888 phosphate D-fructose 6-phosphate = UDP + (spsA)synthase sucrose 6-phosphate (EC 2.4.1.14) sucrose sucrose 6-phosphate +H2O = AmyA phosphorylase sucrose + phosphate (A2022) (EC 3.1.3.24)α-1,4-glucan Linear alpha-glucan = glucose + lyase1,5-anhydro-D-fructose (EC 4.2.2.13) glycogen UDP-glucose + synthase(1,4-alpha-D-glucosyl)n = UDP + (EC 2.4.1.11) (1,4-alpha-D-glucosyl)n +1 1,4-α-glucan transfers a segment of a A1865 branching1,4-alpha-D-glucan chain to a enzyme primary hydroxy group in a (EC2.4.1.18) similar glucan chain

Phototrophic fixation of CO2 is followed by the rapid flux of carboncompounds to the creation and maintenance of biomass and to the storageof retrievable carbon in the form of glycogen, cellulose and/or sucrose.Under medium conditions of sufficient nitrogen, glycogen stores canrepresent 30% of cell mass. Under nitrogen starvation conditions, cellspartition more carbon to glycogen creating stores up to 60% of cellmass. Nitrogen limitation can act as a biological control over carbonflux to glycogen.

Glycogen is a polymer of glucose composed of linear alpha 1,4-linkagesand branched alpha 1,6-linkages. The polymer is insoluble at degree ofpolymerization (DP) greater than about 60,000 and forms intracellulargranules. Glycogen in synthesized in vivo via a pathway originating fromglucose 1-phosphate. Its hydrolysis can proceed through phosphorylationto glucose phosphates; via the internal cleavage of polymer tomaltodextrins; via the successive exo-cleavage to maltose; or via theconcerted hydrolysis of polymer and maltodextrins to maltose andglucose.

In certain aspects, various routes to engineer metabolism to produceglucose biosynthetically are described. For example, glycogen synthesiscan be interrupted, and glucose-1-phosphate or glucose-6-phosphate canbe desphosphorylated. Glucose phosphate could be dephosphorylatedintracellularly via a cloned or endogenous phosphatase or hexokinase andtransported out of the cell via a cloned or endogenous facilitatingcarrier. Alternatively, the glucose phosphate could be transported anddephosphorylated externally. The glucose phosphate could also be useddirectly as a fermentation substrate.

In addition to the above, another mechanism is described to produceglucose biosynthetically. In certain embodiments, the present inventionprovides for cloned genes for glycogen hydrolyzing enzymes to hydrolyzeglycogen to glucose and/or maltose and transport maltose and glucosefrom the cell. Preferred enzymes are set forth below in Table 2. Glucoseis transported out by a glucose/hexose transporter. This alternativeallows the cell to accumulate glycogen naturally but adds enzymeactivities to continuously return it to maltose or glucose units whichcan be collected as a fermentable product.

There are a number of potential enzyme candidates for glycogenhydrolysis. Enzymes are limited in their mechanisms for hydrolysis ofthe 1,4- and 1,6-bonds of the glycogen polymer and complete hydrolysisrequires an ensemble of enzymes. α-amylases perform an endo-attack onlarge polymers of glycogen and hydrolysis results in formation ofshorter, average DP 13, polymers which are attacked in an exo-fashion byglucoamylase to result in glucose product. Neither of the aforementionedenzymes will attack at the 1,6-branches. Therefore, pullulanases andother amylo-1,6-glycosidases, which in nature perform this hydrolysis,are used to completely hydrolyze glycogen to glucose. An alternative isa β-amylase which performs exo-attack on the large polymer ends andresults in release of maltose units. Additionally, there are a number ofpossibilities for enzymatic dephosphorylation of glucose-6-phosphateincluding alkaline or acid phosphatases and kinases. The followingenzymes listed in Table 2 below have activities specific to sugar orsugar polymer dephosphorylation.

TABLE 2 Enzymes for hydrolysis of glycogen Enzyme Enzyme NameClassification No. Function α-amylase EC 3.2.1.1 endohydrolysis of1,4-alpha-D-glucosidic linkages in polysaccharides β-amylase EC 3.2.1.2hydrolysis of 1,4-alpha-D-glucosidic linkages in polysaccharides so asto remove successive maltose units from the non-reducing ends of thechains γ-amylase EC 3.2.1.3 hydrolysis of terminal 1,4-linkedalpha-D-glucose residues successively from non-reducing ends of thechains with release of beta-D-glucose glucoamylase EC 3.2.1.3 hydrolysisof terminal 1,4-linked alpha-D-glucose residues successively fromnon-reducing ends of the chains with release of beta-D-glucoseisoamylase EC 3.2.1.68 hydrolysis of (1−>6)-alpha-D-glucosidic branchlinkages in glycogen, amylopectin and their beta-limit dextrinspullulanase EC 3.2.1.41 hydrolysis of (1−>6)-alpha-D-glucosidic linkagesin pullulan [a linear polymer of alpha-(1−>6)-linked maltotriose units]and in amylopectin and glycogen, and the alpha- and beta-limit dextrinsof amylopectin and glycogen amylomaltase: EC 2.4.1.25; transfers asegment of a 1,4-alpha-D-glucan to a new position in an acceptor, whichmay be glucose or a 1,4-alpha-D-glucan (part of yeast debranchingsystem) amylo-α-1,6- EC 3.2.1.33 debranching enzyme; glucosidasehydrolysis of (1−>6)-alpha-D-glucosidic branch linkages in glycogenphosphorylase limit dextrin phosphorylase EC 2.7.11.19 2 ATP +phosphorylase b = kinase 2 ADP + phosphorylase a phosphorylase EC2.4.1.1 (1,4-alpha-D-glucosyl)n + phosphate = (1,4-alpha-D-glucosyl)n −1 + alpha-D-glucose-1-phosphateTransport/Efflux Gene Products

A number of transport mechanisms are possible. Most bacterial cells havevectorial active transporters to move glucose or maltose into the cell.To accumulate sugars, these mechanisms rely on energy coupling in theform of ATP, proton motive force or gradients of other molecularspecies, e.g., phosphate. Plant chloroplasts have active mechanisms tofacilitate efflux of glucose and maltose to the plant or algalcytoplasm. Accordingly, in certain embodiments, building transportersinto the inner membrane may involve targeting and assembly, andvectoriality of the energy coupling mechanism versus solute flux. Forinstance, maltose efflux pump from chloroplast for maltose transport:MEX1; glucose permeases, low and high Km, glucose:H+ symporter,glucose/fructose permease, general sugar:H+ antiporter for glucosetransport; and glucose 6-phosphate:Pi antiporter,triose-phosphate:phosphate antiporter for glucose-6-phosphate transportare contemplated transport mechanisms of the present invention.

There are natural Chlorella algal strains that secrete maltose andglucose at appreciable rates. These strains are normally endosymbioticand, remarkably, when isolated freshly from their hosts excrete almostall of their photosynthate as extracellular monosaccharide, however,almost invariably, they lose this ability soon after being removed.

A few Chlorella strains can be grown as axenic cultures (˜12 hr doublingtime, 30° C.) and still secrete appreciable fractions (5-40%) of theirphotosynthate almost entirely as either glucose or maltose on nutrientstarvation media (akin to glycogen production upon nitrogen starvation).These excretion rates continue in the dark from intracellular stores ofphotosynthate. Some of the best rates in the literature are described byFischer et al. 179:251-256 (1989); and Brechignac et al., Adv. SpaceRes. 14:79-88 (1994).

In certain embodiments, the above mentioned rates of sugar productionare maintained or, more preferably, exceeded. Operating at a biomassdensity of 15 g/l (˜OD 50), implies a volumetric productivity of˜0.05*15=0.75 g sugar/l/hr.

The fermentation products according to the above aspect of the inventionare sugars, which are exported into the media as a result of carbonfixation during photosynthesis. The sugars can be reabsorbed later andfermented, directly separated, or utilized by a co-cultured organism.This approach has several advantages. First, the total amount of sugarsthe cell can handle is not limited by maximum intracellularconcentrations because the end-product is exported to the media. Second,by removing the sugars from the cell, the equilibria of carbon fixationreactions are pushed towards creating more sugar. Third, duringphotosynthesis, there is no need to push carbon flow towards glycolysis.Fourth, the sugars are potentially less toxic than the fermentationproducts that would be directly produced.

Accordingly, the invention provides cells which produce metabolicsugars, e.g., glucose, through photosynthesis using light, water andCO₂, subsequently converting the sugars into carbon-based products ofinterest in an efficient, sustainable yield. In certain embodiments, thephotosynthetic organisms are genetically modified to producephotosynthetic products such as glucose at amounts greater than 1 mg,100 mg, 500 mg, 1 g, 5 g, 10 g, 20 g, 25 g, 30 g, 35 g, 40 g, 50 g, 100g, 120 g, or 150 g per liter of fermentation medium.

The invention also provides engineered photosynthetic organisms thatproduce other sugars such as sucrose, xylose, pentose, rhamnose, andarabinose according to the same principles. Using such sugars as itsprimary carbon source, the organism can ferment the sugar and producecarbon-based products of interest, e.g., biofuels such as ethanol (see,e.g., Ho et al., Appl Environ Microbiol, 64:1852-1859 (1998), describinguse of glucose and xylose for the producing ethanol from cellulosicbiomass).

Consolidated Photo-Fermentation

The above aspect of the invention is an alternative to directlyproducing final carbon-based product of interest as a result ofphotosynthesis. In this approach, carbon-based products of interestwould be produced by leveraging other organisms that are more amenableto making any one particular product while culturing the photosyntheticorganism for its carbon source. Consequently, fermentation andproduction of carbon-based products of interest can occur separatelyfrom carbon source production in a photobioreactor.

In one aspect, the methods of producing such carbon-based products ofinterest include two steps. The first-step includes using photosyntheticorganisms to convert carbon dioxide to photosynthetic products such asglucose. The second-step is to use the photosynthetic products as acarbon source for cells that produce carbon-based products of interest.In one embodiment, the two-stage approach comprises a photobioreactorcomprising photosynthetic cells; a second reactor comprising cellscapable of fermentation; wherein the photosynthetic cells provides acarbon source such as glucose for cells capable of fermentation toproduce a carbon-based product of interest. The second reactor maycomprise more than one type of microorganism. The resultingcarbons-based products of interest are subsequently separated and/orcollected.

Preferably, the two-steps are combined into a single-step processwhereby the engineered photosynthetic organisms convert light and CO₂directly into glucose and such organisms are capable of producing avariety of carbon-based products of interest.

The present invention also provides methods and compositions forsustained glucose production in photosynthetic organisms wherein theseor other organisms that use the sugars are cultured using light, waterand CO₂ for use as a carbon source to produce carbon-based products ofinterest. In such embodiments, the host cells are capable of secretingthe sugars, such as glucose from within the cell to the culture media incontinuous or fed-batch in a bioreactor.

Certain changes in culture conditions of photosynthetic host cells,e.g., cyanobacteria for the production of sugars can be optimized forgrowth. For example, conditions are optimized for light intensity, lightexposure, time of exposure, diurnal cycle, addition of supplements,nutrients, the rate of recirculation and flow rates that maintain alight to dark ratio. As will be apparent to those skilled in the art,the conditions sufficient to achieve optimum growth will vary dependingupon location, climate, and other environmental factors, such as thediurnal cycle, light intensity and time of exposure to light. Otheradjustments may be required, for example, an organism's ability forcarbon uptake. Increased carbon in the form of CO₂ may be introducedinto a bioreactor by a gas sparger or aeration devices

Advantages of consolidated photo-fermentation include a process wherethere is separation of chemical end products, e.g., glucose, spatialseparation between end products (membranes) and time. Additionally,unlike traditional or cellulosic biomass to biofuels production,pretreatment, saccharification and crop plowing are obviated.

The consolidated photo-fermentation process produces continuousproducts. In preferred embodiments, the process involves direct captureof light to product from engineered front-end organisms to producevarious products without the need to lyse the organisms. For instance,the organisms can utilize 3PGAL in the light to make a desiredfermentation product, e.g., ethanol. In other embodiments, the organismscan accumulate glycogen in the light and metabolize ethanol in the darkto make more fermentation products. Such end products can be readilysecreted as opposed to intracellular products such as oil and cellulose.In yet other embodiments, organisms produce sugars in the light, whichare secreted into the media and such sugars are used in the dark duringfermentation with the same or different organisms or a combination ofboth.

Fermentation Conditions

The production and isolation of carbon-based products of interest can beenhanced by employing specific fermentation techniques. One method formaximizing production while reducing costs is increasing the percentageof the carbon that is converted to hydrocarbon products. During normalcellular lifecycles carbon is used in cellular functions includingproducing lipids, saccharides, proteins, organic acids, and nucleicacids. Reducing the amount of carbon necessary for growth-relatedactivities can increase the efficiency of carbon source conversion tooutput. This can be achieved by first growing microorganisms to adesired density, such as a density achieved at the peak of the log phaseof growth. At such a point, replication checkpoint genes can beharnessed to stop the growth of cells. Specifically, quorum sensingmechanisms [reviewed in Camilli and Bassler, Science 311:1113, (2006);Venturi FEMS Microbio Rev 30:274-291 (2006); and Reading and Sperandio,FEMS Microbiol Lett, 254:1-11, (2006)] can be used to activate genessuch as p53, p21, or other checkpoint genes. Genes that can be activatedto stop cell replication and growth in E. coli include umuDC genes, theover-expression of which stops the progression from stationary phase toexponential growth [Murli et al, J. of Bact., 182:1127, (2000)]. UmuC isa DNA polymerase that can carry out translesion synthesis overnon-coding lesions—the mechanistic basis of most UV and chemicalmutagenesis. The umuDC gene products are used for the process oftranslesion synthesis and also serve as a DNA damage checkpoint. UmuDCgene products include UmuC, UmuD, umuD′, UmuD′₂C, UmuD′₂ and UmUD₂.Simultaneously, the product-producing genes are activated, thusminimizing the need for replication and maintenance pathways to be usedwhile the fatty acid derivative is being made.

In one aspect, the percentage of input carbons converted to hydrocarbonproducts is an efficient and inexpensive process. Using carbon dioxideas the carbon source, the oxygen is released in the form of O₂, leadingto a maximal theoretical metabolic efficiency of ˜34% (w/w) (for fattyacid derived products).

This figure, however, changes for other hydrocarbon products and carbonsources. Typical efficiencies in the literature are ˜<5%. Engineeredmicroorganisms which produce hydrocarbon products can have greater than1, 3, 5, 10, 15, 20, 25, and 30% efficiency. In one examplemicroorganisms will exhibit an efficiency of about 10% to about 25%. Inother examples, such microorganisms will exhibit an efficiency of about25% to about 30%, and in other examples such microorganisms willexhibit >30% efficiency.

In some examples where the final product is released from the cell, acontinuous process can be employed. In this approach, a reactor withorganisms producing fatty acid derivatives can be assembled in multipleways. In one example, a portion of the media is removed and allowed toseparate. Fatty acid derivatives are separated from the aqueous layer,which will in turn, be returned to the fermentation chamber.

In another example, the fermentation chamber will enclose a fermentationthat is undergoing a continuous reduction. In this instance, a stablereductive environment would be created. The electron balance would bemaintained by the release of oxygen. Efforts to augment the NAD/H andNADP/H balance can also facilitate in stabilizing the electron balance.

The availability of intracellular NADPH can be also enhanced byengineering the production host to express an NADH:NADPHtranshydrogenase. The expression of one or more NADH:NADPHtranshydrogenase converts the NADH produced in glycolysis to NADPH whichenhances the production of fatty acid derivatives.

For large-scale product production, the engineered microorganisms aregrown in 10 L, 100 L or larger batches, fermented and induced to expressdesired products based on the specific genes encoded in plasmids asappropriate. Cells harboring engineered nucleic acids to over-express orattenuate gene products are incubated from a 500 mL seed culture for 10L fermentations (5 L for 100 L fermentations) in LB media (glycerolfree) at 37° C. shaken at >200 rpm until cultures reached a desired OD(typically 16 hours) incubated with kanamycin, ampicillin or the like.Media is treated with continuously supplemented to maintain a 25 mMsodium proprionate at a suitable pH of about 8.0 to activate theengineered in gene systems for production as well as to stop cellularproliferation. Media is continuously supplemented with carbon dioxide.Aliquots of no more than 10% of the total cell volume are removed eachhour and allowed to sit unaggitated so as to allow the hydrocarbonproduct to rise to the surface and undergo a spontaneous phaseseparation. The hydrocarbon component is then collected and the aqueousphase returned to the reaction chamber. The reaction chamber is operatedcontinuously. For wax ester production, subsequent to isolation, the waxesters are washed briefly in 1 M HCl to split the ester bond, andreturned to pH 7 with extensive washing with distilled water.

Production and Release of Fatty Alcohol from Production Host

Also disclosed herein is a system for continuously producing andexporting hydrocarbons out of recombinant host microorganisms via atransport protein. Many transport and efflux proteins serve to excrete alarge variety of compounds and can be evolved to be selective for aparticular type of fatty acid. Thus, in some embodiments an exogenousnucleic acid sequence encoding an ABC transporter will be functionallyexpressed by the recombinant host microorganism, so that themicroorganism exports the fatty acid into the culture medium. In oneexample, the ABC transporter is an ABC transporter from Caenorhabditiselegans, Arabidopsis thalania, Alkaligenes eutrophus or Rhodococcuserythropolis (locus AAN73268). In another example, the ABC transporteris an ABC transporter chosen from CER5 (locuses AtI g51500 or AY734542),AtMRPS, AmiS2 and AtPGP1. In some examples, the ABC transporter is CER5.In yet another example, the CER5 gene is from Arabidopsis (locuses AtIg51500, AY734542, At3 g21090 and At Ig51460).

The transport protein, for example, can also be an efflux proteinselected from: AcrAB, ToIC and AcrEF from E. coli, or tlll618, H11619and U10139 from Thermosynechococcus elongatus BP-I.

In addition, the transport protein can be, for example, a fatty acidtransport protein (FATP) selected from Drosophila melanogaster,Caenorhabditis elegans, Mycobacterium tuberculosis or Saccharomycescerevisiae or any one of the mammalian FATPs. The FATPs can additionallybe resynthesized with the membranous regions reversed in order to invertthe direction of substrate flow. Specifically, the sequences of aminoacids composing the hydrophilic domains (or membrane domains) of theprotein can be inverted while maintaining the same codons for eachparticular amino acid. The identification of these regions is well knownin the art.

Production hosts can also be selected for their endogenous ability torelease fatty acids. The efficiency of product production and releaseinto the fermentation media can be expressed as a ratio of intracellularproduct to extracellular product. In some examples the ratio can be 5:1,4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, or 1:5.

Processing & Separation

The carbon-based products produced by the carbon dioxide fixingorganisms during fermentation can be separated from the fermentationmedia. Known techniques for separating fatty acid derivatives fromaqueous media can be employed. One exemplary separation process providedherein is a two-phase (bi-phasic) separation process. This processinvolves fermenting the genetically-engineered production hosts underconditions sufficient to produce for example, a fatty acid, allowing thefatty acid to collect in an organic phase and separating the organicphase from the aqueous fermentation media. This method can be practicedin both a batch and continuous fermentation setting.

Bi-phasic separation uses the relative immisciblity of fatty acid tofacilitate separation. A skilled artisan will appreciate that bychoosing a fermentation media and the organic phase such that the fattyacid derivative being produced has a high log P value, even at very lowconcentrations the fatty acid will separate into the organic phase inthe fermentation vessel.

When producing fatty acids by the methods described herein, suchproducts will be relatively immiscible in the fermentation media, aswell as in the cytoplasm. Therefore, the fatty acid will collect in anorganic phase either intracellularly or extracellularly. The collectionof the products in an organic phase will lessen the impact of the fattyacid derivative on cellular function and allows the production host toproduce more product.

The fatty alcohols, fatty acid esters, waxes, and hydrocarbons producedas described herein allow for the production of homogeneous compoundswith respect to other compounds wherein at least 50%, 60%, 70%, 80%,90%, or 95% of the fatty alcohols, fatty acid esters, waxes andhydrocarbons produced have carbon chain lengths that vary by less than 4carbons, or less than 2 carbons. These compounds can also be produced sothat they have a relatively uniform degree of saturation with respect toother compounds, for example at least 50%, 60%, 70%, 80%, 90%, or 95% ofthe fatty alcohols, fatty acid esters, hydrocarbons and waxes are mono-,di-, or tri-unsaturated.

Pathways Associated with Production of Isoprenoids

There are two known biosynthetic pathways that synthesize isopentenylpyrophosphate (“IPP”) and its isomer, dimethylallyl pyrophosphate(“DMAPP”). Eukaryotes other than plants use the mevalonate-dependent(“MEV”) isoprenoid pathway exclusively to convert acetyl-coenzyme A(“acetyl-CoA”) to IPP, which is subsequently isomerized to DMAPP.Prokaryotes, with some exceptions, use the mevalonate-independent ordeoxyxylulose 5-phosphate (“DXP”) pathway to produce IPP and DMAPPseparately through a branch point. In general, plants use both the MEVand DXP pathways for IPP synthesis.

MEV Pathway: In general, the pathway comprises six steps. In the firststep, two molecules of acetyl-coenzyme A are enzymatically combined toform acetoacetyl-CoA. An enzyme known to catalyze this step is, forexample, acetyl-CoA thiolase. Examples include without limitation NC000913 REGION: 232413 L.2325315; E. coli, D49362; Paracoccusdenitrificans, and L20428; S. cerevisiae.

In the second step of the MEV pathway, acetoacetyl-CoA is enzymaticallycondensed with another molecule of acetyl-CoA to form3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). An enzyme known to catalyzethis step is, for example, HMG-CoA synthase. Examples include withoutlimitation NC_(—)001 145 complement 19061 . . . 20536; S. cerevisiae,X96617; S. cerevisiae, X83882; A. thaliana, AB037907; Kitasatosporagriseola, BT007302; H. sapiens, and NC_(—)002758, Locus tag SAV2546,GeneID 1 122571; S. aureus.

In the third step, HMG-CoA is enzymatically converted to mevalonate.HMG-CoA reductase is an example of n enzyme known to catalyze this step.Examples from various organisms include, without limitation,NM_(—)206548; D. melanogaster, NC_(—)002758, Locus tag SAV2545, GeneID1122570; S. aureus, NM_(—)204485; Gallus gallus, AB015627; Streptomycessp. KO 3988, AF542543; Nicotiana attenuata, AB037907; Kitasatosporagriseola, AX128213, providing the sequence encoding a truncated HMGR; S.cerevisiae, and NC_(—)001 145: complement 115734 . . . 1 18898; S.cerevisiae.

In the fourth step, mevalonate is enzymatically phosphorylated to formmevalonate 5-phosphate. An enzyme known to catalyze this step is, forexample, mevalonate kinase. Examples include without limitation L77688;A. thaliana, and X55875; S. cerevisiae.

In the fifth step, a second phosphate group is enzymatically added tomevalonate 5-phosphate to form mevalonate 5-pyrophosphate. An enzymeknown to catalyze this step is, for example, phosphomevalonate kinase.Examples include without limitation AF429385; Hevea brasiliensis,NM_(—)006556; H. sapiens, and NC_(—)001 145 complement 712315 . . .713670; S. cerevisiae.

In the sixth step, mevalonate 5-pyrophosphate is enzymatically convertedinto IPP. An enzyme known to catalyze this step is, for example,mevalonate pyrophosphate decarboxylase. Examples include withoutlimitation X97557; S. cerevisiae, AF290095; E. faecium, and U49260; H.sapiens.

If IPP is to be converted to DMAPP using the mevalonate pathway, then aseventh step is required. An enzyme known to catalyze this step is, forexample, IPP isomerase. Examples include without limitation NC 000913,3031087, 3031635; E. coli, and AF082326; Haematococcus pluvialis.

DXP Pathway: In general, the DXP pathway comprises seven steps In thefirst step, pyruvate is condensed with D-glyceraldehyde 3-phosphate tomake 1-deoxy-D-xylulose-5-phosphate. An enzyme known to catalyze thisstep is, for example, 1-deoxy-D-xylulose-5-phosphate synthase. Examplesinclude without limitation AF035440; E. coli, NC_(—)002947, locus tagPP0527; P. putida KT2440, CP000026, locus tag SPA2301; Salmonellaenterica Paratyphi, see ATCC 9150, NC_(—)007493, locus tag RSP_(—)0254;Rhodobacter sphaeroides 2.4.1, NC_(—)005296, locus tag RPA0952;Rhodopseudomonas palustris CGA009, (NC_(—)004556, locus tag PD 1293;Xylellafastidiosa Temeculal, and NC_(—)003076, locus tag AT5G11380; A.thaliana.

In the second step, 1-deoxy-D-xylulose-5-phosphate is converted to2C-methyl-D-erythritol-4-phosphate. An enzyme known to catalyze thisstep is, for example, 1-deoxy-D-xylulose-5-phosphate reductoisomerase.Examples include without limitation AB013300; E. coli, AF148852; A.thaliana, NC_(—)002947, locus tag PP 1597; Pseudomonas putida KT2440,AL939124, locus tag SCO5694; Streptomyces coelicolor A3(2),(NC_(—)007493), locus tag RSP_(—)2709; Rhodobacter sphaeroides 2.4.1,and NC_(—)007492, locus tag Pfl_(—)1107; Pseudomonas fluorescens PfO-1.

In the third step, 2C-methyl-D-erythritol-4-phosphate is converted to4-diphosphocytidyl-2C-methyl-D-erythritol. An enzyme known to catalyzethis step is, for example, 4-diphosphocytidyl-2C-methyl-D-erythritolsynthase. Examples include without limitation AF230736; E. coli,NC_(—)007493, locus_tag RSP_(—)2835; Rhodobacter sphaeroides 2.4.1,NC_(—)003071, locus tag AT2G02500; A. thaliana, and NC 002947, locus_tagPP 1614; P. putida KT2440).

In the fourth step, 4-diphosphocytidyl-2C-methyl-D-erythritol isconverted to 4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate. Anenzyme known to catalyze this step is, for example,4-diphosphocytidyl-2C-methyl-D-erythritol kinase. Examples includewithout limitation AF216300; E. coli and NC_(—)007493, locus_tagRSP_(—)1779; Rhodobacter sphaeroides 2.4.1).

In the fifth step, 4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphateis converted to 2C-methyl-D-erythritol 2,4-cyclodiphosphate. An enzymeknown to catalyze this step is, for example, 2C-methyl-D-erythritol2,4-cyclodiphosphate synthase. Examples include without limitationAF230738; E. coli, NC_(—)007493, locus_tag RSP_(—)6071; Rhodobactersphaeroides 2.4.1, and NC_(—)002947, locus tag PP1 618; P. putidaKT2440.

In the sixth step, 2C-methyl-D-erythritol 2,4-cyclodiphosphate isconverted to 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate. An enzymeknown to catalyze this step is, for example,1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase. Examplesinclude without limitation AY033515; E. coli, NC_(—)002947, locus_tagPP0853; P. putida KT2440, and NC007493, locus_tag RSP_(—)2982;Rhodobacter sphaeroides 2.4.1.

In the seventh step, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate isconverted into either IPP or its isomer, DMAPP. An enzyme known tocatalyze this step is, for example, isopentyl/dimethylallyl diphosphatesynthase. Examples include without limitation AY062212; E. coli andNC_(—)002947, locus_tag PP0606; P. putida KT2440.

Isoprenoid Production

Any suitable host cell as described herein may be used in the practiceof the present invention. In one embodiment, the host cell is agenetically modified host microorganism in which nucleic acid moleculeshave been inserted, deleted or modified (i.e., mutated, e.g., byinsertion, deletion, substitution, and/or inversion of nucleotides), toeither produce the desired isoprenoid compound or starting material, orto increase yields of the desired isoprenoid compound or or startingmaterial. In another embodiment, the host cell is capable of being grownin liquid growth medium.

Provided herein is a method to produce isoprenoids in carbon dioxidefixing hosts engineered with the isopentenyl pyrophosphate pathwayenzymes. Some examples of isoprenoids include: hemiterpenes (derivedfrom 1 isoprene unit) such as isoprene; monoterpenes (derived from 2isoprene units) such as myrcene; sesquiterpenes (derived from 3 isopreneunits) such as amorpha-4,11-diene; diterpenes (derived from fourisoprene units) such as taxadiene; triterpenes (derived from 6 isopreneunits) such as squalene; tetraterpenes (derived from 8 isoprenoids) suchas β-carotene; and polyterpenes (derived from more than 8 isopreneunits) such as polyisoprene. The production of isoprenoids is alsodescribed in some detail in the published PCT applications WO2007/139925and WO/2007/140339.

In one aspect, a host cell producing isoprenoid involves the steps ofselecting a host cell that is capable or can be modified to produce anenzymatic pathway for making isopentenyl pyrophosphate wherein the allof the pathway enzymes are under expression control sequences; andculturing the host cells in a medium under suitable conditions forgrowth. In some embodiments, the pathway is the mevalonate pathway. Inother embodiments, the pathway is the DXP pathway.

In some embodiments, “cross talk” (or interference) between the hostcells own metabolic processes and those processes involved with theproduction of IPP are minimized or eliminated entirely. For example,cross talk is minimized or eliminated entirely when the hostmicroorganism relies exclusively on the DXP pathway for synthesizingIPP, and a MEV pathway is introduced to provide additional IPP. Such ahost organism would not be equipped to alter the expression of the MEVpathway enzymes or process the intermediates associated with the MEVpathway. Organisms that rely exclusively or predominately on the DXPpathway include, for example, E. coli.

In some embodiments, the host cell produces IPP via the MEV pathway,either exclusively or in combination with the DXP pathway. In otherembodiments, a host cell's DXP pathway is functionally disabled so thatthe host cell produces IPP exclusively through a heterologouslyintroduced MEV pathway. The DXP pathway can be functionally disabled bydisabling gene expression or inactivating the function of one or more ofthe DXP pathway enzymes.

In some embodiments, the host cell produces IPP via the DXP pathway,either exclusively or in combination with the MEV pathway. In otherembodiments, a host cell's MEV pathway is functionally disabled so thatthe host cell produces IPP exclusively through a heterologouslyintroduced DXP pathway. The MEV pathway can be functionally disabled bydisabling gene expression or inactivating the function of one or more ofthe MEV pathway enzymes.

In yet another embodiment, a method for producing an isoprenoid orisoprenoid precursor comprises the steps of (i) performing afermentation reaction comprising a fermentation medium and a pluralityof genetically modified carbon dioxide fixing host cells that producethe isoprenoid under conditions such that (a) the fermentation medium iskept at a temperature lower than that which would provide for a maximumspecific growth rate of said host cells; (b) the fermentation mediumcomprises a carbon dioxide; and/or (c) the fermentation medium comprisesa nitrogen source present in an amount that is lower than that whichwould provide for a maximum specific growth rate of the host cells; (ii)recovering the isoprenoid produced under one or more conditions setforth in (a) through (c). In one aspect, the isoprenoid is producedunder at least two of the conditions set forth in (a) through (c). Inanother aspect, the isoprenoid is produced under all of the conditionsset forth in (a) through (c).

In a further aspect of the present invention, compositions and methodsare provided for a robust production of isoprenoids by the use ofisopentenyl pyrophosphate pathway enzymes that are under the control ofat least one heterologous regulator or fermentation conditions, eitheralone or in combination.

In yet another aspect, a method for producing an isoprenoid involves thesteps of (a) selecting or obtaining genetically modified carbon dioxidefixing host cells that comprise an enzymatic pathway for makingisopentenyl pyrophosphate wherein all of the pathway enzymes are undercontrol of at least one heterologous transcriptional regulator; and (b)culturing the host cells in a medium under conditions that aresuboptimal as compared to conditions that would provide for a maximumspecific growth rate for the host cells. In some embodiments, thepathway is the mevalonate pathway. In other embodiments, the pathway isthe DXP pathway. In other embodiments, the pathway enzymes are underexpression control sequences.

In some embodiments, the pathway comprises a nucleic acid sequenceencoding a mevalonate pathway enzyme from a prokaryote having anendogenous mevalonate pathway. Exemplary prokaryotes having anendogenous mevalonate pathway include but are not limited to the genusEnterococcus, the genus Pseudomonas, and the genus Staphylococcus. Inone embodiment, the mevalonate pathway enzyme is selected fromacetyl-CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, and mevalonatekinase. In another embodiment, the heterologous nucleic acid sequenceencodes a Class II HMG-CoA reductase. In other embodiments, host cellssuch as cyanobacteria are engineered to heterologously express amevalonate pathway.

In some embodiments, the amount of the isoprenoid compound produced bythe host cell is at least 30% by volume based on the total volume of thebiofuel.

In another embodiment, the host cells are cultured in a medium whereinthe nutrient and/or temperature level is maintained at a level belowthat which would provide for the maximum specific growth rate for thehost cells. In another embodiment, the host cells are cultured in amedium where the carbon source is maintained at a level to provide forless than about 90%, 75%, 50%, 25%, 10%, or anywhere between 90% and 10%of the maximum specific growth rate. In another embodiment, the hostcells are cultured in a medium where the nitrogen source is maintainedat a level to provide for less than about 90%, 75%, 50%, 25%, 10%, oranywhere between 90% and 10% of the maximum specific growth rate Inanother embodiment, the host cells are cultured in a medium where thetemperature is maintained at a level to provide for less than about 90%,75%, 50%, 25%, 10% or anywhere between 90% and 10% of the maximumspecific growth rate. In another embodiment, the medium temperature ismaintained at least about 2° C., 4° C., 5° C., 6° C., 8° C., 10° C., 15°C., or 20° C. below the temperature that would provide for the maximumspecific growth rate.

Fuel Compositions

The above compositions produced by the carbon dioxide fixing organisms,carbon-based products, e.g., ethanol, fatty acids, alkanes, isoprenoidscan be used as fuel. For example, using the methods described hereinfuels comprising relatively homogeneous fatty acid derivatives that havedesired fuel qualities can be produced. Such fuels can be characterizedby carbon fingerprinting, their lack of impurities when compared topetroleum-derived fuels or bio-diesel derived from triglycerides and,moreover, the fatty-acid-based fuels can be combined with other fuels orfuel additives to produce fuels having desired properties.

Similar to the fuels from fatty acids, the present invention encompassesa fuel composition comprising a fuel component and a bioengineered C₅isoprenoid compound.

In another aspect, the invention encompasses a fuel composition producedby preparing 3-methyl-3-buten-1-ol using a carbon dioxide fixingmicroorganism, and incorporating the 3-methyl-3-buten-1-ol in a fuel.

In another aspect, the invention encompasses a fuel composition producedby preparing 3-methyl-2-buten-1-ol using a microorganism, andincorporating the 3-methyl-2-buten-1-ol in a fuel.

In another aspect, the invention encompasses a fuel composition producedby preparing 3-methyl-3-buten-1-ol using a microorganism, preparingisoamyl alcohol from the 3-methyl-3-buten-1-ol, and incorporating theisoamyl alcohol in a fuel.

In another aspect, the invention encompasses a fuel composition producedby preparing 3-methyl-2-buten-1-ol using a microorganism, preparingisoamyl alcohol from the 3-methyl-2-buten-1-ol, and incorporating theisoamyl alcohol in a fuel.

In some embodiments, the recombinant host cell is modified to increasean enzymatic conversion of isopentenyl pyrophosphate (IPP),dimethylallyl pyrophosphate (DMAPP), or a combination thereof to anisopentenol.

In certain embodiments, the biofuel comprises 3-methyl-3-buten-1-ol,3-methyl-2-buten-1-ol, 3-methyl-1-butanol or a combination thereof. Infurther embodiments, the amount of 3-methyl-3-buten-1-ol,3-methyl-2-buten-1-ol or 3-methyl-1-butanol is at least about 2%.

Methods of preparing the isoprenoid compound using one or moremicroorganisms are described in Example 20. In certain embodiments, thefuel composition is produced by preparing 3-methyl-3-buten-1-ol usingone or more microorganisms, preparing 3-methyl-1-butanol from3-methyl-3-buten-1-ol, and incorporating the 3-methyl-1-butanol in thefuel composition. In other embodiments, the fuel composition is producedby preparing 3-methyl-2-buten-1-ol using one or more microorganisms,preparing 3-methyl-1-butanol from 3-methyl-2-buten-1-ol, andincorporating the 3-methyl-1-butanol in the fuel composition.

Impurities

In certain aspects, carbon-based products, e.g., ethanol, fatty acids,alkanes, isoprenoids produced herein contain fewer impurities than arenormally associated with biofuels derived from triglycerides, such asfuels derived from vegetable oils and fats. For instance, crude fattyacid biofuels described herein (prior to mixing the fatty acidderivative with other fuels such as petrochemical diesel or bio-diesel)can contain less glycerol (or glycerin) than bio-fuels made fromtriglycerides, crude biofuel can contain less free alcohol (i.e.,alcohol that is used to create the ester) than biodiesel made fromtriglycerides. Biofuels characteristically have a low concentration ofsulfur compared to petroleum-derived diesel.

In one aspect, the crude fatty acid biofuels described herein (prior tomixing the fatty acid derivative with other fuels such as traditionalfuels) can contain less transesterification catalyst than petrochemicaldiesel or bio-diesel. Preferably, the fatty acid derivative can containless than about 2%, 1.5%, 1.0%, 0.5%, 0.3%, 0.1%, 0.05%, or 0% of atransesterification catalyst, or an impurity resulting from atransesterification catalyst, glycerol, free alcohol or sulfur.Transesterification catalysts include, for example, hydroxide catalystssuch as NaOH, KOH, LiOH, and acidic catalysts, such as mineral acidcatalysts and Lewis acid catalysts. Catalysts and impurities resultingfrom transesterification catalysts include, without limitation, tin,lead, mercury, cadmium, zinc, titanium, zirconium, hafnium, boron,aluminum, phosphorus, arsenic, antimony, bismuth, calcium, magnesium,strontium, uranium, potassium, sodium, lithium, and combinationsthereof.

The differences in composition of gasoline may require that, in order toproduce a uniform product, blending of the products from severalcomponent streams may be necessary. The properties of each stream mayvary considerably, significantly affecting the product gasoline. Theblending process is relatively straightforward, but the determination ofthe amount of each component to include in a blend is much moredifficult.

In certain embodiments, the fuel composition disclosed herein is free orsubstantially free of a second alcohol wherein the second alcohol is not3-methyl-3-buten-1-ol, 3-methyl-2-buten-1-ol or a combination thereof.In further embodiments, the second alcohol is methanol, ethanol,n-propanol, iso-propanol, n-butanol, iso-butanol, tert-butanol,n-pentanol, sec-pentanol, tert-pentanol, n-hexanol, iso-hexanol,sec-hexanol, tert-hexanol, heptanols, octanols, nonanols, decanols or acombination thereof. In some embodiments, the fuel composition disclosedherein is free or substantially free of an aromatic compound. In otherembodiments, the fuel composition disclosed herein is free orsubstantially free of an alkylamine, fatty acid ester or fatty acidsalt.

In certain embodiments, the fuel composition disclosed herein furthercomprises a petroleum-based fuel in an amount from 1% to 95% by volume,based on the total volume of the fuel composition. In some embodiments,the petroleum-based fuel is gasoline. In further embodiments, the C₅isoprenoid compound is present in an amount from about 1% to about 5% byvolume, from about 1% to about 10% by volume, from about 1% to about12.5% by volume, from about 2.5% to about 12.5% by volume, or from about5% to about 12.5% by volume, based on the total volume of the fuelcomposition.

Additives

Generally, fuel additives are used to enhance the performance of a fuelor engine. For example, fuel additives can be used to alter thefreezing/gelling point, cloud point, lubricity, viscosity, oxidativestability, ignition quality, octane level, and flash point. A skilledartisan will recognize that the fatty acids described herein can bemixed with other fuels such as bio-diesel derived from triglycerides,various alcohols such as ethanol and butanol, and petroleum-derivedproducts such as gasoline. In some examples, a fatty acid, such as C16:1ethyl ester or C18:1 ethyl ester, is produced which has a low gel point.This low gel point fatty acid derivative is mixed with bio-diesel madefrom triglycerides to lessen the overall gelling point of the fuel.Similarly, a fatty acid derivative such as C16:1 ethyl ester or C18:1ethyl ester can be mixed with petroleum-derived diesel to provide amixture that is at least and often greater than 5% biodiesel. In someexamples, the mixture includes at least 20% or greater of the fattyacid.

For example, a biofuel composition can be made that includes at leastabout 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% of a fatty acidthat includes a carbon chain that is 8:0, 10:0, 12:0, 14:0, 14:1, 16:0,16:1, 18:0, 18:1, 18:2, 18:3, 20:0, 20:1, 20:2, 20:3, 22:0, 22:1 or22:3. Such biofuel compositions can additionally include at least oneadditive selected from a cloud point-lowering additive that can lowerthe cloud point to less than about 5° C., or 0° C., a surfactant, or amicroemulsion, at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%or 80%, 85%, 90%, or 95% diesel fuel from triglycerides,petroleum-derived gasoline or diesel fuel from petroleum.

In some embodiments, the biofuel further comprises a petroleum-basedfuel, a fuel additive or a combination thereof. In further embodiments,the petroleum-based fuel is a gasoline, jet fuel, kerosene, diesel fuelor a combination thereof.

The amount of the C₅ isoprenoid compound or a derivative thereof in thefuel composition disclosed herein may be from 0.5% to 99%, from 0.5% to98%, from 1% to 97%, from 1% to 96%, from 2% to 95%, from 2% to 90%,from 3% to 85%, or from 5% to 80%, based on the total amount of the fuelcomposition. In certain embodiments, the C₅ isoprenoid or derivativethereof is a C₅ cyclic hydrocarbon. The amount of the C₅ cyclichydrocarbon is more than 1%, more than 2%, more than 3%, more than 4%,more than 5%, more than 10%, more than 15%, more than 20%, more than25%, more than 30%, more than 35%, more than 40%, more than 45%, morethan 50%, more than 55%, more than 60%, more than 65%, more than 70%,more than 75%, more than 80%, more than 85%, more than 90% or more than95%, based on the total amount of the fuel composition. In someembodiments, the amount is in wt. % based on the total weight of thefuel composition. In other embodiments, the amount is in vol. % based onthe total volume of the fuel composition. In certain embodiments, thefuel composition is a gasoline fuel composition.

The amount of the petroleum-based fuel component in the fuel compositiondisclosed herein may be from 0.1% to 99%, from 1% to 95%, from 2% to90%, from 3% to 85%, from 5% to 80%, from 5% to 70%, from 5% to 60%, orfrom 5% to 50%, based on the total amount of the fuel composition. Incertain embodiments, the amount of the petroleum-based fuel component isless than 95%, less than 90%, less than 85%, less than 75%, less than70%, less than 65%, less than 60%, less than 55%, less than 50%, lessthan 45%, less than 40%, less than 35%, less than 30%, less than 25%,less than 20%, less than 15%, less than 10%, less than 5%, less than 4%,less than 3%, less than 2%, less than 1% or less than 0.5%, based on thetotal amount of the fuel composition. In some embodiments, the amount isin wt. % based on the total weight of the fuel composition. In otherembodiments, the amount is in vol. % based on the total volume of thefuel composition. In certain embodiments, the fuel composition is agasoline fuel composition.

In certain embodiments, the fuel additive in the fuel compositiondisclosed herein is selected from the group consisting of oxygenates,antioxidants, thermal stability improvers, cetane improvers,stabilizers, cold flow improvers, combustion improvers, anti-foams,anti-haze additives, corrosion inhibitors, lubricity improvers, icinginhibitors, injector cleanliness additives, smoke suppressants, dragreducing additives, metal deactivators, dispersants, detergents,demulsifiers, dyes, markers, static dissipaters, biocides andcombinations thereof. In further embodiments, the amount of the fueladditive is from about 0.1% to about 20% by weight or volume, based onthe total weight or volume of the fuel composition.

The most common jet fuel is a kerosene/paraffin oil-based fuelclassified as Jet A-1, which is produced to an internationallystandardized set of specifications. In the United States only, a versionof Jet A-1 known as Jet A is also used. Another jet fuel that iscommonly used in civilian aviation is called Jet B. Jet B is a lighterfuel in the naptha-kerosene region that is used for its enhancedcold-weather performance. Jet A, Jet A-1 and Jet B are specified in ASTMSpecification D. 1655-68. Alternatively, jet fuels are classified bymilitaries around the world with a different system of JP numbers. Someare almost identical to their civilian counterparts and differ only bythe amounts of a few additives. For example, Jet A-1 is similar to JP-8and Jet B is similar to JP-4. Alternatively, jet fuels can also beclassified as kerosene or naphtha-type. Some non-limiting examples ofkerosene-type jet fuels include Jet A, Jet A1, JP-5 and JP-8. Somenon-limiting examples of naphtha-type jet fuels include Jet B and JP-4.Jet A is used in the United States while most of the rest of the worlduses Jet A-1. An important difference between Jet A and Jet A-1 is themaximum freezing point. Jet A-1 has a lower maximum freezing temperatureof −47° C. while Jet A has a maximum freezing temperature of −40° C.Like Jet A-1, Jet A has a fairly high flash point of minimum 38° C.,with an autoignition temperature of 210° C.

The amount of each of the conventional fuel additives in the fuelcomposition disclosed herein may be from 0.1% to less than 50%, from0.2% to 40%, from 0.3% to 30%, from 0.4% to 20%, from 0.5% to 15% orfrom 0.5% to 10%, based on the total amount of the fuel composition. Incertain embodiments, the amount of each of the conventional fueladditives is less than 50%, less than 45%, less than 40%, less than 35%,less than 30%, less than 25%, less than 20%, less than 15%, less than10%, less than 5%, less than 4%, less than 3%, less than 2%, less than1% or less than 0.5%, based on the total amount of the fuel composition.In some embodiments, the amount is in wt % based on the total weight ofthe fuel composition. In other embodiments, the amount is in volume %based on the total volume of the fuel composition.

Some conventional fuel additives have been described in “Gasoline:Additives, Emissions, and Performance” by Society of AutomotiveEngineers, SAE International, 1995 (ISBN: 1560916451), which isincorporated herein by reference. Further, the following U.S. patentsdisclose various fuel additives that can be employed in embodiments ofthe invention as additives: U.S. Pat. Nos. 6,054,420; 6,051,039;5,997,593; 5,997,592; 5,993,498; 5,968,211; 5,958,089; 5,931,977;5,891,203; 5,882,364; 5,880,075; 5,880,072; 5,855,629; 5,853,436;5,743,922; 5,630,852; 5,529,706; 5,505,867; 5,492,544; 5,490,864;5,484,462; 5,321,172; and 5,284,492. The disclosures of all of thepreceding U.S. patents are incorporated by reference herein in theirentirety for all purposes.

Any oxygenate that increases the weight % of oxygen in the fuelcomposition disclosed herein can be used. Generally, oxygenates arecombustible liquids comprises carbon, hydrogen and oxygen that can becategorized into two classes of organic compounds, i.e., alcohols andethers. Some non-limiting examples of suitable oxygenates includeethanol, methyl tertiary-butyl ether (MTBE), tertiary-amyl methyl ether(TAME), and ethyl tertiary-butyl ether (ETBE).

Any lubricity improver that increases the fuel lubricity can be used. Insome embodiments, one or more lubricity improvers are mixed with thefuel composition disclosed herein. Typically, the concentration of thelubricity improver in the fuel falls in the range of from 1 to 50,000ppm, preferably about 10 to 20,000 ppm, and more preferably from 25 to10,000 ppm. Some non-limiting examples of lubricity improver includeesters of fatty acids.

Any combustion improver that can increase the mass burning rate of thefuel composition disclosed herein can be used. Some non-limitingexamples of combustion improvers include ferrocene(dicyclopentadienyliron), iron-based combustion improvers (e.g., TURBOTECT™ ER-18 fromTurbotect (USA) Inc., Tomball, Tex.), barium-based combustion improvers,cerium-based combustion improvers, and iron and magnesium-basedcombustion improvers (e.g., TURBOTECT™ 703 from Turbotect (USA) Inc.,Tomball, Tex.). The combustion improver may be present in the fuelcomposition at a concentration of about 0.001 to 1 wt %, based on thetotal weight of the fuel composition, and in one embodiment from 0.01 to1% by weight.

In some embodiments, the fuel compositions comprise an antioxidant. Anyantioxidant that can prevent the formation of gum depositions on fuelsystem components caused by oxidation of fuels in storage and/or inhibitthe formation of peroxide compounds in certain fuel compositions can beused herein. The antioxidant may be present in the fuel composition at aconcentration of about 0.001 to 5 wt %, based on the total weight of thefuel composition, and in one embodiment from 0.01 to 1% by weight.

In other embodiments, the fuel compositions comprise a staticdissipater. Static dissipaters reduce the effects of static electricitygenerated by movement of fuel through high flow-rate fuel transfersystems. The static dissipater may be present in the fuel composition ata concentration of about 0.001 to 5 wt %, based on the total weight ofthe fuel composition, and in one embodiment from 0.01 to 1% by weight.

In further embodiments, the fuel compositions comprise a corrosioninhibitor. Corrosion inhibitors protect ferrous metals in fuel-handlingsystems such as pipelines, and fuel storage tanks, from corrosion. Incircumstances where additional lubricity is desired, corrosioninhibitors that also improve the lubricating properties of thecomposition can be used. The corrosion inhibitor may be present in thefuel composition at a concentration of about 0.001 to 5 wt %, based onthe total weight of the fuel composition, and in one embodiment from0.01 to 1% by weight.

In certain embodiments, the fuel composition comprises a fuel systemicing inhibitor (also referred to as an anti-icing additive). Fuelsystem icing inhibitors reduce the freezing point of water precipitatedfrom jet fuels due to cooling at high altitudes and prevent theformation of ice crystals which restrict the flow of fuel to the engine.Certain fuel system icing inhibitors can also act as a biocide. The fuelsystem icing inhibitor may be present in the fuel composition at aconcentration of about 0.001 to 5 wt %, based on the total weight of thefuel composition, and in one embodiment from 0.01 to 1% by weight.

In another set of embodiments, the fuel compositions further comprise abiocide. Biocides are used to combat microbial growth in the fuelcomposition. The biocide may be present in the fuel composition at aconcentration of about 0.001 to 5 wt %, based on the total weight of thefuel composition, and in one embodiment from 0.01 to 1% by weight.

In another set of embodiments, the fuel composition further comprises ametal deactivator. Metal deactivators suppress the catalytic effect somemetals, particularly copper, have on fuel oxidation. The metaldeactivator may be present in the fuel composition at a concentration ofabout 0.001 to 5 wt %, based on the total weight of the fuelcomposition, and in one embodiment from 0.01 to 1% by weight.

In another set of embodiments, the fuel composition further comprises athermal stability improver. Thermal stability improvers are use toinhibit deposit formation in the high-temperature areas of the aircraftfuel system. The thermal stability improver may be present in the fuelcomposition at a concentration of about 0.001 to 5 wt %, based on thetotal weight of the fuel composition, and in one embodiment from 0.01 to1% by weight.

Volatility is an important property of gasoline and is a necessity toensure engine starting in cold weather. In winter, volatility is raisedand the flash point is lowered by adding the more volatile butanes andpentanes. To prevent vapor lock in warm weather, the amounts of the morevolatile constituents are reduced to produce mixtures that will notvaporize in the fuel lines.

Detection and Analysis

Generally, the products of interest produced from the “solarbiofactories” described herein can be analyzed by any of the standardanalytical methods, e.g., gas chromatography (GC), mass spectrometry(MS) gas chromatography-mass spectrometry (GCMS), and liquidchromatography-mass spectrometry (LCMS), high performance liquidchromatography (HPLC), capillary electrophoresis, Matrix-Assisted LaserDesorption Ionization time-of-flight mass spectrometry (MALDI-TOF MS),nuclear magnetic resonance (NMR), near-infrared (NIR) spectroscopy,viscometry [Knothe et al., Am. Chem. Soc. Symp. Series, 666:172-208(1997)], titration for determining free fatty acids [Komers et al.,Fett/Lipid 99(2):52-54 (1997)], enzymatic methods [Bailer et al., J.Anal. Chem. 340(3):186 (1991)], physical property-based methods, wetchemical methods, etc.

Carbon Fingerprinting

Biologically-produced carbon-based products, e.g., ethanol, fatty acids,alkanes, isoprenoids, represent a new commodity for fuels, such asalcohols, diesel and gasoline. Such biofuels have not been producedusing biomass but use CO₂ as its carbon source. These new fuels may bedistinguishable from fuels derived form petrochemical carbon on thebasis of dual carbon-isotopic fingerprinting. Such products,derivatives, and mixtures thereof may be completely distinguished fromtheir petrochemical derived counterparts on the basis of ¹⁴C (fM) anddual carbon-isotopic fingerprinting, indicating new compositions ofmatter.

There are three naturally occurring isotopes of carbon: ¹²C, ¹³C, and¹⁴C. These isotopes occur in above-ground total carbon at fractions of0.989, 0.011, and 10⁻¹², respectively. The isotopes ¹²C and ¹³C arestable, while ¹⁴C decays naturally to ¹⁴N, a beta particle, and ananti-neutrino in a process with a half-life of 5730 years. The isotope¹⁴C originates in the atmosphere, due primarily to neutron bombardmentof ¹⁴N caused ultimately by cosmic radiation. Because of its relativelyshort half-life (in geologic terms), ¹⁴C occurs at extremely low levelsin fossil carbon. Over the course of 1 million years without exposure tothe atmosphere, just 1 part in 10⁵⁰ will remain ¹⁴C.

The ¹³C:¹²C ratio varies slightly but measurably among natural carbonsources. Generally these differences are expressed as deviations fromthe ¹³C:¹²C ratio in a standard material. The international standard forcarbon is Pee Dee Belemnite, a form of limestone found in SouthCarolina, with a ¹³C fraction of 0.0112372. For a carbon source a, thedeviation of the ¹³C:¹²C ratio from that of Pee Dee Belemnite isexpressed as:

δ_(a)=(R_(a)/R_(s))−1, where R_(a)=¹³C:¹²C ratio in the natural source,and R_(s)=¹³C:¹²C ratio in Pee Dee Belemnite, the standard.

For convenience, δ_(a) is expressed in parts per thousand, or ‰. Anegative value of δ_(a) shows a bias toward ¹²C over ¹³C as compared toPee Dee Belemnite. Table 3 shows δ_(a) and ¹⁴C fraction for severalnatural sources of carbon.

TABLE 3 ¹³C:¹²C variations in natural carbon sources Source −δ_(a) (‰)References Underground coal 32.5 Farquhar et al. Fossil fuels 26Farquhar et al. Ocean DIC*   0-1.5 Goericke et al., Ivlev AtmosphericCO2 6-8 Ivlev, Farquhar et al. Freshwater DIC*  6-14 Dettman et al. PeeDee Belemnite 0 Ivlev *DIC = dissolved inorganic carbon

Biological processes often discriminate among carbon isotopes. Thenatural abundance of ¹⁴C is very small, and hence discrimination for oragainst ¹⁴C is difficult to measure. Biological discrimination between¹³C and ¹²C, however, is well-documented. For a biological product p, wecan define similar quantities to those above:

δ_(p)=(R_(p)/R_(s))−1, where R_(p)=¹³C:¹²C ratio in the biologicalproduct, and R_(s)=¹³C:¹²C ratio in Pee Dee Belemnite, the standard.

Table 4 shows measured deviations in the ¹³C:¹²C ratio for somebiological products.

TABLE 4 ¹³C:¹²C variations in selected biological products Product−δ_(p)(‰) −D(‰)* References Plant sugar/starch from 18-28   10-20 Ivlevatmospheric CO₂ Cyanobacterial biomass 18-31 16.5-31 Goericke et al.,from marine DIC Sakata et al. Cyanobacterial lipid 39-40 37.5-40 Sakataet al. from marine DIC Algal lipid from 17-28 15.5-28 Goericke et al.,marine DIC Abelseon et al. Algal biomass from 17-36   3-30 Marty et al.freshwater DIC E. coli lipid from 15-27 near 0 Monson et al. plant sugarCyanobacterial lipid 63.5-66   37.5-40 — from fossil carbonCyanobacterial biomass 42.5-57   16.5-31 — from fossil carbon *D =discrimination by a biological process in its utilization of ¹²C vs. ¹³C(see text)

Table 2 introduces a new quantity, D. This is the discrimination by abiological process in its utilization of ¹²C vs. ¹³C. We define D asfollows: D=(R_(p)/R_(a))−1.

This quantity is very similar to δ_(a) and δ_(p), except we now comparethe biological product directly to the carbon source rather than to astandard. Using D, we can combine the bias effects of a carbon sourceand a biological process to obtain the bias of the biological product ascompared to the standard. Solving for δ_(p), we obtain:δ_(p)=(D)(δ_(a))+D+δ_(a), and, because (D)(δ_(a)) is generally verysmall compared to the other terms, δ_(p)≈δ_(a)+D.

For a biological product having a production process with a known D, wemay therefore estimate δ_(p) by summing δ_(a) and D. We assume that Doperates irrespective of the carbon source.

This has been done in Table 2 for cyanobacterial lipid and biomassproduced from fossil carbon. As shown in the Tables above,cyanobacterial products made from fossil carbon (in the form of, forexample, flue gas or other emissions) will have a higher δ_(p) thanthose of comparable biological products made from other sources,distinguishing them on the basis of composition of matter from theseother biological products. In addition, any product derived solely fromfossil carbon will have a negligible fraction of ¹⁴C, while productsmade from above-ground carbon will have a ¹⁴C fraction of approximately10⁻¹².

Accordingly, in certain aspects, the invention provides variouscarbon-based products of interest characterized as −δ_(p)(‰) of about63.5 to about 66 and −D(‰) of about 37.5 to about 40.

REFERENCES

-   1. Goericke, R., Montoya, J. P., and Fry, B. Physiology of isotopic    fractionation in algae and cyanobacteria. Chapter 9 in “Stable    Isotopes in Ecology and Environmental Science”,-   By K. Lajtha and R. H. Michener, Blackwell Publishing, 1994.-   2. Monson, K. D. and Hayes, J. M. Biosynthetic control of the    natural abundance of carbon 13 at specific positions within fatty    acids in Escherichia coli. J. Biol. Chem. 255:11435-41 (1980).-   3. Abelseon, P. H. and Hoering, T. C. Carbon isotope fractionation    in formation of amino acids by photosynthetic organisms. Proc. Natl.    Acad. Sci. 47:623-32 (1961).-   4. Sakata, S., Hayes, J. M., McTaggart, A. R., Evans, R. A.,    Leckrone, K. J., and Togasaki, R. K. Carbon isotopic fractionation    associated with lipid biosynthesis by a cyanobacterium: relevance    for interpretation of biomarker records. Geochim. Cosmochim. Acta    61:5379-89 (1997).-   5. Ivlev, A. A. Carbon isotope effects (13C/12C) in biological    systems. Separation Sci. Technol. 36:1819-1914 (20010).-   6. Farquhar, G. D., Ehleringer, J. R., and Hubick, K. T. Carbon    isotope discrimination and photosynthesis. Annu. Rev. Plant Physiol.    Plant Mol. Biol. 40:503-37 (1989).-   7. Marty, J. and Planas, D. Comparison of methods to determine algal    δ¹³C in freshwater. Limnol. Oceanogr.: Methods 6:51-63 (2008).-   8. Dettman, D. L., Reische, A. K., and K. C. Lohmann. Controls on    the stable isotope composition of seasonal growth bands in    aragonitic fresh-water bivalves (unionidae). Geochim. Cosmochim.    Acta 63:1049-1057 (1999).

All publications and patent documents cited herein are herebyincorporated by reference in their entirety for all purposes to the sameextent as if each were so individually denoted.

EXAMPLES

The examples below are provided herein for illustrative purposes and arenot intended to be restrictive.

Example 1 Plasmid Construction for Synechococcus sp. PCC 7002

Construction of pJB5: The pJB5 base plasmid was designed as an emptyexpression vector for recombination into Synechococcus sp. PCC 7002. Tworegions of homology, the Upstream Homology Region (UHR) and theDownstream Homology Region were designed to flank the construct. These500 bp regions of homology correspond to positions 3301-3800 and3801-4300 (Genbank Accession NC_(—)005025) for UHR and DHR respectively.The aadA promoter, gene sequence, and terminator were designed to conferspectinomycin and streptomycin resistance to the integrated construct.For expression, pJB5 was designed with the aph2 kanamycin resistancecassette promoter and ribosome binding site (RBS). Downstream of thispromoter and RBS, we designed and inserted the restriction endonucleaserecognition site for NdeI and EcoRI, as well as the sites for XhoI,BamHI, SpeI and PacI. Following the EcoRI site, the natural terminatorfrom the alcohol dehydrogenase gene from Zymomonas mobilis (adhII)terminator was included. Convenient xbaI restriction sites flank the UHRand the DHR allowing cleavage of the DNA intended for recombination fromthe rest of the vector. pJB5 was constructed by contract synthesis fromDNA2.0 (Menlo Park, Calif.).

Construction of pJB5-PdcAdhII The pyruvate decarboxylase (pdc) andalcohol dehydrogenase (adhII) genes were cloned into the pJB5 plasmidwith the following procedure. The pdc-adhII genes from Zymomonas mobilis(Genbank: DD161475, M15394) were designed with an NdeI site replacingthe start of the pdc coding region. Following the pdc gene, we designedtwo restriction endonuclease sites (XhoI and BamHI). Next, the adhIIsequence was designed in whole subsequent to the restriction sites, andfinally, the natural adhII terminator was included as well, downstreamof an inserted EcoRI site. This construct was constructed by contractsynthesis from DNA2.0 (Menlo Park, Calif.) and was inserted byrestriction digest with NdeI and EcoRI (New England Biolabs; Ipswitch,Mass.) on both pJB5 and the insert followed by ligation with a QuickLigation Kit (New England Biolabs; Ipswitch, Mass.). The ligatedconstruct was transformed into The NEB 5-alpha F′Iq Competent E. coli(High Efficiency) (New England Biolabs: Ipswitch, Mass.).

pJB5-PdcAdhII(TS): The pyruvate decarboxylase (pdc) from Zymobacterpalmae (GenBank: AF474145) and alcohol dehydrogenase TS42 (adhII) genesas described in Rellos et al. (1998) “Thermostable variants of Zymomonasmobilis alcohol dehydrogenase obtained using PCR-mediated randommutagenesis” Protein Expr Purif 12:61-61) were cloned into the pJB5plasmid with the following procedure. These genes were designed with anNdeI site replacing the start of the pdc coding region. Following thepdc gene and prior to the adhII gene, a gap is present which includesXhoI and BamHI sites to allow promoters to be inserted later (totallength of gap: 39 bp) and the original RBS for adhII from Z. mobilis.The adhII (Z. mobilis) gene has the original terminator presentafterwards, in which an EcoRI site has been placed between the adhIIgene and the terminator. Following the terminator, SpeI and PacI sitesare present for cloning. This construct was constructed by contractsynthesis from DNA2.0 (Menlo Park, Calif.) and was inserted byrestriction digest with NdeI and EcoRI (New England Biolabs; Ipswitch,Mass.) on both pJB5 and the insert followed by ligation with a QuickLigation Kit (New England Biolabs; Ipswitch, Mass.). The ligatedconstruct was transformed into The NEB 5-alpha F′Iq Competent E. coli(High Efficiency) (New England Biolabs; Ipswitch, Mass.).

pJB5-Pdc: The pyruvate decarboxylase (pdc) gene was cloned into the pJB5plasmid with the following procedure. The pJB5-PdcAdhII construct fromExample 2, was digested with BamHI and EcoRI (New England Biolabs;Ipswitch, Mass.). The incompatible 5′ and 3′ DNA overhangs were removedusing the Quick Blunting Kit (New England Biolabs, MA), and then ligatedusing the Quick Ligation Kit (New England Biolabs; Ipswitch, Mass.).

pJB5-AdhII: The alcohol dehydrogenase (adhII) gene was cloned into thepJB5 plasmid with the following procedure. The pJB5-PdcAdhII constructfrom Example 2, was digested with NdeI and BamHI (New England Biolabs;Ipswitch, Mass.). The incompatible 5′ and 3′ DNA overhangs were removedusing the Quick Blunting Kit (New England Biolabs, MA), and then ligatedusing the Quick Ligation Kit (New England Biolabs; Ipswitch, Mass.).

pJB5-metE (E. coli): The Vitamin B12 independent methionine synthase(metE) gene from E. coli (Genbank: NP_(—)418273.1), was cloned into thepJB5 plasmid by the following procedure. A construct was synthesized bycontract synthesis by DNA2.0 (Menlo Park, Calif.) to include an NdeIsite to replacing the start of the metE gene, and an EcoRI site at theend of the gene. This construct was inserted by restriction digest withNdeI and EcoRI (New England Biolabs; Ipswitch, Mass.) on both pJB5 andthe insert followed by ligation with a Quick Ligation Kit (New EnglandBiolabs; Ipswitch, Mass.). The ligated construct was transformed intoThe NEB 5-alpha F′Iq Competent E. coli (High Efficiency) (New EnglandBiolabs: Ipswitch, Mass.).

pJB5-metE (T. elongates BP-1): The Vitamin B12-independent methioninesynthase (metE) gene from Thermosynechococcus elongates BP-1 (Genbank:NP_(—)681881), was cloned into the pJB5 plasmid by the followingprocedure. A construct was synthesized by contract synthesis by DNA2.0(Menlo Park, Calif.) to include an NdeI site to replacethe start of themetE gene, and an EcoRI site at the end of the gene. This construct wasinserted by restriction digest with NdeI and EcoRI (New England Biolabs;Ipswitch, Mass.) on both pJB5 and the insert followed by ligation with aQuick Ligation Kit (New England Biolabs; Ipswitch, Mass.). The ligatedconstruct was transformed into The NEB 5-alpha F′Iq Competent E. coli(High Efficiency) (New England Biolabs: Ipswitch, Mass.).

Example 2 Plasmid Construction for Thermosynechococcus elongatus BP-1

Thermosynechococcus elongatus BP-1 is selected as another exemplary CO₂fixing production host and is modified by engineered nucleic acids tofunctionally delete certain genes and/or to express, overexpress certaingenes.

Four plasmids (pJB18, pJB19, pJB20, and pJB21), all derivatives of pJB5,were constructed to permit homologous recombination into four differentloci in the Thermosynechococcus elongatus BP-1 genome. Specifically, the0.5 kb upstream homology (UH) and downstream homology (DH) regions usedfor Synechococcus sp. PCC 7002 homologous recombination in pJB5 werereplaced by the following approximately 2.5 kb T. elongatus BP-1(Accession NC_(—)004113) regions: coordinates 831908-834231 (UH) and834232-836607 (DH) genome for pJB18, 454847-457252 (UH) and457252-459740 (DH) for pJB19, 481310-483712 (UH) and 483709-486109 (DH)for pJB20, and 787356-789654 (UH) 791080-793494 (DH) for pJB21. Thefirst three homology regions are based on integration sites TS1, TS3,and TS4 described in Onai K. et al. (2004). “Natural transformation ofthe thermophilic cyanobacterium Thermosynechococcus elongatus BP-1: asimple and efficient method for gene transfer.” Mol. Gen. Genomics 271:50-59. The last is designed to delete completely the glgA open readingframe, encoding glycogen synthase: The purpose of this deletion is tominimize competing fixed carbon flux towards glycogen once theethanol-producing genes are integrated into the chromosome.

All T. elongatus BP-1 homology regions were generated by PCR usingPhusion™ Hot Start High-Fidelity DNA Polymerase (Developed &Manufactured By Finnzymes Oy. Distributed by New England Biolabs,Ipswitch, Mass.) according to manufacturer's instructions. The UHforward PCR primer has a 5′-terminal SbfI restriction site, the UHreverse PCR primer a 5′-terminal NotI restriction site, the DH forwardPCR primer a 5′-terminal AscI restriction site, and the DH reverse PCRprimer a 5′-terminal FseI restriction site. For pJB 18, pJB 19, pJB20,and pJB21, the UH region is first inserted into pJB5 via restrictiondigestion with SbfI and NotI (New England Biolabs; Ipswitch, Mass.) ofboth vector and PCR-generated insert, followed by with a Quick LigationKit (New England Biolabs; Ipswitch, Mass.). The ligated construct istransformed into NEB 5-alpha Competent E. coli (High Efficiency) (NewEngland Biolabs: Ipswitch, Mass.). The sequence of the UH region in pHB5is validated by contract sequencing with GENEWIZ (South Plainfield,N.J.). For pJB18, pJB19, pJB20, and pJB21, the DH region is theninserted into the pJB5-UH region construct exactly as done for the UHregion, except that restriction enzymes AscI and FseI are used (NewEngland Biolabs; Ipswitch, Mass.). DH regions are sequence confirmed bycontract sequencing with GENEWIZ (South Plainfield, N.J.).

Into each of pJB 18, pJB 19, pJB20, and pJB21, two different versions ofthe pyruvate decarboxylase (pdc)/alcohol dehydrogenase (adhII) operonsare cloned, creating a set of eight plasmids ready for integration intothe T. elongatus BP-1 genome. In each case, the selectable marker is thepJB5 aadA gene encoding resistance to spectinomycin and streptomycin.The first version of the operon comprises the pdc and adhII genes fromZymomonas mobilis (Genbank: DD161475, M15394) and is designed with anNdeI site covering the start codon of the pdc coding sequence. Followingthe pdc gene in order are: an XhoI restriction site, a BamHI restrictionsite, the adhII coding sequence, the natural Zymomonas mobilis adhIIterminator, and finally an EcoRI restriction site. The second version ofthe operon, designed to encode relatively more thermostable versions ofpyruvate decarboxylase and alcohol dehydrogenase, comprised the pdc genefrom Zymobacter palmae (GenBank: AF474145) and the adhII mutant TS42described in Rellos et al., Protein Expr. Purif., 12:61-61 (1998), andis otherwise identical to the first construct in all other ways. Bothconstructs are made by contract synthesis from DNA2.0 (Menlo Park,Calif.) and are inserted by restriction digest with NdeI and EcoRI (NewEngland Biolabs; Ipswitch, Mass.) into pJB18, pJB19, pJB20, and pJB21,followed by ligation with a Quick Ligation Kit (New England Biolabs;Ipswitch, Mass.). In this way eight pdc-adhII operon plasmids areconstructed: pJB22, pJB23, pJB24, and pJB25 containing operon version 1,based on pJB18, pJB19, pJB20, and pJB21, respectively, and pJB26, pJB27,pJB28, and pJB29 containing operon version 2, based on pJB18, pJB19,pJB20, and pJB21, respectively.

In plasmids pJB22, pJB23, pJB24, pJB25, pJB26, pJB27, pJB28, and pJB29,the pdc-adhII operon is expressed by the constitutive P_(aphII)promoter, which is flanked by unique NotI and NdeI restriction sites.These sites permit other constitutive promoters to be cloned in, in lieuof the P_(aphII) promoter, in case that promoter does not affordsufficient expression of the operon when integrated into the genome ofT. elongatus BP-1. Separate plasmids are constructed (pJB9, pJB10,pJB11, pJB12, pJB13, pJB14, pJB15, pJB16, and pJB17), all made bycontract synthesis e.g., DNA2.0 (Menlo Park, Calif.), each bearing oneof nine candidate alternative constitutive promoters flanked by NotI andNdeI sites so they can replace the P_(aphII) promoter by standardcloning methods. Seven of those promoters are native T. elongatus BP-1promoters, corresponding to the upstream sequence of the followinggenes: cpcC, apcA, tsr2142, psaA, rbcL, hsp33, and trnE_UUC and two areE. coli-type promoters: P_(tac) (as described in De Boer et al., ProcNatl Acad USA 80:21-25 (1983)) and the synthetic P_(EM7) promoter.

Example 3 Engineered Microorganisms Producing Ethanol

Genetically Modified Synechococcus sp. PCC 7002: Each of the constructsas described in Example 1 was integrated onto the genome ofSynechococcus sp. PCC 7002 using the following protocol. Synechococcus7002 was grown for 48 h from colonies in an incubated shaker flask at30° C. at 1% CO₂ to an OD₇₃₀ of 11n A⁺ medium described in Frigaard N Uet al. (2004) “Gene inactivation in the cyanobacterium Synechococcus sp.PCC 7002 and the green sulfur bacterium Chlorobium tepidum using invitro-made DNA constructs and natural transformation” Methods Mol Biol274:325-340. 500 μL of culture was added to a test-tube with 30 μL of1-5 μg of DNA prepped from a Qiagen Qiaprep Spin Miniprep Kit (Valencia,Calif.) for each construct. Cells were incubated bubbling in 1% CO₂ atapproximately 1 bubble every 2 seconds for 4 hours. 200 μL of cells wereplated on A⁺ medium plates with 1.5% agarose and grown at 30° C. for twodays in low light. 10 μg/mL of spectinomycin was underplayed on theplates. Resistant colonies were visible in 7-10 days.

Strain Construction and Expression of Moorella sp. HUC22-1 AdhA: Thesequence for Moorella sp. HUC22-1 AdhA has been shown to be an NADPutilizing alcohol dehydrogenase that is also thermostable andpreferential for the reduction of acetaldehyde [Inokuma et al., Arch.Microbiol., 188:37-45 (2007)]. While the sequence has not beenpublished, the amino acid similarity to AdhIV from Moorellathermoacetica (Accession Number: ABC20211) was 100%. The nucleic acidsequence of AdhIV from Moorella thermoacetica (Accession Number:CP000232) was codon optimized for expression and constructed by DNA 2.0and designated as SEQ ID NO: 1 (the encoded amino acid is SEQ ID NO: 2).The sequence is flanked with CTCGAGTTGGATCC on the 5′ end, which encodesthe Xho and BamHI restriction sites, and on the 3′ end withTTTCAAAACAGGAATTC on the 3′ end (similar to pJB5-3) which contains anEcoRI site for the purposes of cloning into expression vectors.

The Moorella adhA was then cloned downstream of two pyruvatedecarboxylase genes, one from Zymomonas mobilis (Accession number:AAV89984) and one from Zymobacter palmae (Accession Number: AAM49566) toform the expression plasmids pJB 136 and pJB 133, respectively. Ascontrols, expression plasmids were constructed for the Z. mobilispyruvate decarboxylase gene with the Z. mobilis adhII (Accession Number:YP_(—)163331), and the Z. palmae pyruvate decarboxylase gene with animproved thermotolerant adhII TS42 [Rellos et al., Protein Expressionand Purification, 12:61-66 (1998)] to form pJB5-3 and pJB5-4respectively.

The plasmids pJB5-3, pJB5-4, pJB133, pJB136 were cloned intoSynechococcus sp. PCC 7002 (JCC1) using standard procedures anddesignated as JCC136, JCC137, JCC445, JCC446 respectively (Table 5).

TABLE 5 Integration Pyruvate Alcohol Host Construct decarboxylaseDehydrogenase JCC136 pJB5-3 Z. mobilis pdc Z. mobilis adhII JCC137pJB5-4 Z. palmae pdc Z. mobilis adhII TS42 JCC445 pJB133 Z. mobilis pdcMoorella adhA JCC446 pJB136 Z. mobilis pdc Moorella adhA

JCC1, JCC136, JCC137, JCC445, JCC446 were grown on A⁺ media plates (1.5%agar) with 100 ug/mL spectinomycin for transgenic strains. A singlecolony was grown in 10 mL A+ with 100 ug/mL spectinomycin in a test tubeimmersed in a 37 C bath with 1% CO₂ bubbled through. Cultures were grownto OD_(730nm) 5.0 or higher (Molecular Devices Spectramax M2e;previously determined that an OD_(730nm) of 1 is equal to ˜0.3 g CDW),and then spun down (21,000 RCF, 20 C, 5 min), resuspended in fresh A+media to original concentration, and then appropriately back-diluted toOD_(730nm) 0.2 in 25 mL A⁺ in a baffled 125 mL shaker flask.Approximately 1 mL of culture was taken for each time point (0, 6, 24,48, 72 hours post-dilution; 6 hour time point not plotted for timespacing reasons), OD_(730nm) was recorded (appropriately diluted to givereading between 0.04 and 0.4, which was previously determined to be mostaccurate range on the Spectramax M2e). Samples were immediately spundown at 4 C for 10 min at 21,000 RCF. Supernatant was placed in a newtube, and frozen at −80 C until ready for analysis.

Supernatant of each time point was analyzed for ethanol and acetaldehydeby use of an Agilent 7890 Gas Chromatograph equipped with a headspaceanalyzer and a flame ionization detector (Agilent) using a J&WScientific DB-ALC1 (Catalog Number: 123-9134; length: 30 m, InnerDiameter, 0.320 mm, Film Thickness: 1.80 um). 100 uL of each samples wassubjected to headspace analysis. Controls were measured for A+ alone,and as well as from serial dilution of standards for ethanol andacetaldehyde obtained from Sigma to obtain a calibration curve.

To measure the optical densities, ethanol and acetaldehydeconcentrations, cultures were backdiluted from OD_(730nm) 5 or greaterto a starting OD_(730nm) and timepoints were taken at 0, 24, 48, and 72hours post-dilution.

Optical densities of wildtype and various transgenic Synechococcus sp.cultures are shown (FIG. 11). The graph shows plots of OD_(730nm)measurements at each timepoint. Resulting OD measurements are shownTable 6.

TABLE 6 OD (730 nm) Time 0 24 48 72 JCC1 0.257 2.19 5.7 10.1 JCC1360.259 2.26 5.06 7.6 JCC137 0.263 2.265 5.02 8 JCC445 0.246 1.52 4.166.35 JCC446 0.227 1.71 4.52 6.95

Ethanol concentrations of cultures in the supernatant are plottedshowing increased ethanol concentrations with respect to time in variousin transgenic Synechococcus species cultures (FIG. 12). Notably, higherethanol concentration was measured in JCC445, the strain transformedwith Moorella adhA at 72 hours (Table 7).

TABLE 7 EtOH (mg/L) 0 24 48 72 JCC1 0 0 1.704728 5.880188 JCC136 063.00976 140.7334 252.8226 JCC137 0 72.02925 137.0422 256.4378 JCC445 014.03474 153.5205 296.761 JCC446 0 16.06255 125.6418 249.6592

Additionally, decreased acetaldehdye concentrations were observed atvarious timepoints in the strains transformed with Moorella adhA (FIG.13 and Table 8).

TABLE 8 Acetaldehdye (mg/L) 0 24 48 72 JCC1 0 0 0.411352 0.362828 JCC1360 14.20144 34.95365 36.49536 JCC137 0 19.80197 36.05125 35.83849 JCC4450 9.455919 10.82248 13.57957 JCC446 0 8.368128 9.070718 12.32025

At later timepoints, the strains transformed with Moorella adhA (JCC445and JCC446) show marked increases in the ratio of ethanol toacetaldehyde as compared to the Z. mobilis based alcohol dehdyrogenasesas shown in cultures over time (FIG. 14 and Table 9).

TABLE 9 EtOH/Acetaldehdye 0 24 48 72 JCC1 N/A N/A 4.144204 16.20655JCC136 N/A 4.436856 4.026287 6.927526 JCC137 N/A 3.637479 3.8013167.155373 JCC445 N/A 1.484228 14.18532 21.85348 JCC446 N/A 1.91949113.85137 20.26414

FIG. 15 depicts ethanol to OD_(730nm) ratios of cultures over time.Plotted are the ratios of ethanol concentration to OD_(730nm) in thesupernatant at each timepoint. The ratio of the strains transformed withZ. mobilis adh (JCC136 and JCC137) quickly arrive at a steady statewhereas, the ratio of the strains transformed with Moorella adhAconstructs (JCC445 and JCC446) increase over time (Table 10).

TABLE 10 EtOH/OD (mg/L/OD) 0 24 48 72 JCC1 0 0 0.299075 0.582197 JCC1360 27.88043 27.81293 33.26613 JCC137 0 31.80099 27.29924 32.05472 JCC4450 9.233379 36.90395 46.73402 JCC446 0 9.393303 27.79687 35.92218

Genetically Modified Thermosynechococcus elongatus BP-1: From Example 2,pJB22, pJB23, pJB24, pJB25, pJB26, pJB27, pJB28, and pJB29 areintegrated into the chromosome of T. elongatus BP-1 by homologousrecombination using the transformation method detailed in Onai K. et al.(2004). “Natural transformation of the thermophilic cyanobacteriumThermosynechococcus elongatus BP-1: a simple and efficient method forgene transfer.” Mol. Gen. Genomics 271: 50-59. The selection antibioticsused are spectinomycin plus streptomycin.

Example 4 Engineered Microorganisms Producing Butanol

The enzyme beta-ketothiolase (R. eutropha phaA) (EC 2.3.1.16) converts 2acetyl-CoA to acetoacetyl-CoA and CoA. Acetoacetyl-CoA reductase (R.eutropha phaB) (EC 1.1.1.36) generates 3-hydroxybutyryl-CoA fromacetoacetyl-CoA and NADPH. Enoyl-CoA hydratase (E. coli maoC) (EC 4.2.1.{17,55}) generates crotonyl-CoA from 3-hydroxybutyryl-CoA. Butyryl-CoAdehydrogenase (C. acetobutylicum bcd) (EC 1.3.99.2) generatesbutyryl-CoA and NAD(P)H from crotonyl-CoA. Butyrate CoA-transferase (R.eutropha pct) (EC 2.8.3.1) generates butyrate and acetyl-CoA frombutyryl-CoA and acetate. Aldehyde dehydrogenase (E. coli adhE) (EC1.2.1. {3,4}) generates butanal from butyrate and NADH. Alcoholdehydrogenase (E. coli adhE) (EC 1.1.1. {1,2}) generates 1-butanol frombutanal and NADH, NADPH. Production of 1-butanol is conferred by theengineered host cell by expression of the above enzyme activities.

Example 5 Alkane Flux Increasers

At least one of the following enzyme activities are selected andmodified in the host organism. Acetyl-CoA carboxylase (E. coli accABCD)(EC 6.4.1.2, AAN73296) converts acetyl-CoA and CO₂ to malonyl-CoA.Acyl-CoA synthase (E. coli fadD) (EC 2.3.1.86) converts fatty acid andCoA to acyl-CoA. The enzymes TGL2 and LipA (S. cerevisiaetriacylglycerides lipase) (EC 3.1.1.3, AN CAA98876) producestriacylglycerides from fatty acids and glycerol. Lipase (S. cerevisiaeLipA) (EC 3.1.1.3, CAA89087) also produces triacylglycerides from fattyacids and glycerol. and also functions as a suppressor of fabA. Mutationof E. coli K12 plsB D311E (AAC77011) removes limitation on the pool ofacyl-CoA in the host organism. E. coli fabR (NP_(—)418398), a repressorof fatty acid biosynthesis, is deleted for increased unsaturated fattyacid production (Zhang et al., J. Biol. Chem. 277:pp. 15558, 2002).

Example 6 Specific Length Fatty Acid Production

To produce a specific carbon chain length at least one of the followingenzyme activities is modified in the host organism. Thioesterases (EC3.1.2.14) generate acyl-ACP from fatty acid and ACP. The enzyme E. colitesA (AAC73596, P0ADA1) is usually knocked down for an alternate fattyacid C-18:1 thioesterase. One or more of the following is expressed orattenuated depending on desired fatty acid length production: A.thaliana fatA (NP 189147, NP 193041) Bradyrhizobium japonicum fatA, aC-18:1 thioesterase (CAC39106); Cuphea hookeriana fatA, C-18:1thioesterase (AAC72883); Arabidopsis thaliana fatB, C-16:1 thioesterase(CAA85388); Cuphea hookeriana fatB2, C-8:0 to C-10:0 thioesterase(Q39513); Cuphea hookeriana fatB3 C-14:0 to C-16:0 thioesterase(AAC49269; Cinnamonum camphorum fatB C-14:0 thioesterase (Q39473);Umbellularia california fatB C-12:0 thioesterase (Q41635).

Example 7 Unsaturated Fatty Acid Increase

Overexpression of E. coli FabM (DAA05501) may increase unsaturated fattyacid production (trans-2, cis-3-decenoyl-ACP isomerase). Controlledexpression of S. pneumoniae fabK (NP_(—)357969) may also increase inunsaturated fatty acid production (trans-2-enoyl-ACP reductase II).Additionally, E. coli fabI (NP_(—)415804) (trans-2-enoyl-ACP reductase)is attenuated or deleted for increased unsaturated fatty acidproduction. Overexpression of fabB resulted in the production of asignificant percentage of unsaturated fatty acids (de Mendoza et al., J.Biol. Chem., 258:2098-101 (1983)).

Example 8 Unsaturated Fatty Acid Ester

Overexpression of E. coli sfa (AAN79592, AAC44390)-suppressor of fabA,E. coli fabB (EC 3.2.1.41, BAA16180) (B-ketoacyl-ACP synthase I), secGnull mutant suppressors (cold shock proteins) such as E. coli gnsA(ABD18647.1) and E. coli gnsB (AAC74076.1) may increase production ofunsaturated fatty acids. Genes similar to E. coli fabF (YP_(—)852193)are attenuated to increase percentage of C16:1 produced.

Example 9 Conversion of Fatty Aldehyde to Alkane

Decarbonylases convert fatty aldehyde to alkane and CO. For example, A.thaliana cer1 (NP_(—)171723) or Oryza sativa cer1 (AAD29719) isexpressed.

Example 10 Conversion of Fatty Alcohol to Alkane

Terminal alcohol oxidoreducase, e.g., Vibrio furnissii M1 may convertfatty alcohol to alkane acyl terminal alcohol and NADPH to alkanes.

Example 11 Branched Production Alkanes

Step 1 involves expression of a branched-chain amino acidaminotransferase such as E. coli ilvE (EC2.6.1.42, YP_(—)026247),Lactococcus lactis ilvE (EC 2.6.1.42, AAF24406), Pseudomonas putida ilvE(EC 2.6.1.42, NP_(—)745648), Streptomyces coelicolor ilvE (EC 2.6.1.42,NP_(—)629657).

Step 2 involves expression of oxidative decarboyxlation of α-ketoacidsto branched chain acyl-CoA, such as Streptomyces coelicolor bkdA1 (EC1.2.4.4, NP_(—)628006) E1α (decarboxylase component), S. coelicolorbkdB2 (EC 1.2.4.4, NP_(—)628005) E1β (decarboxylase component), S.coelicolor bkdA3 (EC 1.2.4.4, NP_(—)638004) E2 (dihydrolipoyltransacylase); or S. coelicolor bkdA2 (EC 1.2.4.4, NP_(—)733618) E1α(decarboxylase component), S. coelicolor bkdB2 (EC 1.2.4.4,NP_(—)628019) E1β (decarboxylase component), S. coelicolor bkdC2 (EC1.2.4.4, NP_(—)628018) E2 (dihydrolipoyl transacylase); or S.avermitilis bkdA (EC 1.2.4.4, BAC72074) E1α (decarboxylase component),S. avermitilis bkdB (EC 1.2.4.4, BAC72075) E1β (decarboxylasecomponent), S. avermitilis bkdC (EC 1.2.4.4, BAC72076) E2 (dihydrolipoyltransacylase); S. avermitilis bkdF (EC1.2.4.4, BAC72088) E1α(decarboxylase component), S. avermitilis bkdG (EC 1.2.4.4, BAC72089)E1β (decarboxylase component), S. avermitilis bkdH (EC 1.2.4.4,BAC72090) E2 (dihydrolipoyl transacylase); B. subtilis bkdAA (EC1.2.4.4, NP_(—)390288) E1α (decarboxylase component), B. subtilis bkdAB(EC 1.2.4.4, NP_(—)390288) E1β (decarboxylase component), B. subtilisbkdB (EC 1.2.4.4, NP_(—)390288) E2 (dihydrolipoyl transacylase); or P.putida bkdA1 (EC 1.2.4.4, AAA65614) E1α (decarboxylase component), P.putida bkdA2 (EC 1.2.4.4, AAA65615) E13 (decarboxylase component), P.putida bkdC (EC 1.2.4.4, AAA65617) E2 (dihydrolipoyl transacylase); andE. coli Ipd (EC 1.8.1.4, NP_(—)414658) E3 (dihydrolipoyl dehydrogenase).

If the native fatty acid synthase cannot use the branched acyl-CoAs,fabH (EC 2.3.1.41) β-ketoacyl-ACP (acyl carrier protein) synthase IIIwith branched chain acyl CoA specificity can be expressed WITH ACP.Another approach is to express, with ACP, FabF (EC 2.3.1.41)β-ketoacyl-ACP synthase II with branched chain acyl CoA specificity.

Alternatively, fabH is expressed with bkd genes (EC 1.2.4.4.) (Denoya etal. J. Bacteriol 177:pp. 3504, 1995), which consist of E1α/β(decarboxylase), E2 (dihydrolipoyl transacylase) and E3 (dihydrolipoyldehydrogenase) subunits, which are similar to pyruvate andα-ketoglutarate dehydrogenase complexes.

To form branched chain acyl-CoA one or more of the following genes areexpressed: Streptomyces coelicolor fabH1 NP_(—)626634; S. coelicolor ACPNP_(—)626635; S. coelicolor fabF NP_(—)626636; Streptomyces avermitilisfabH3 NP_(—)823466; S. avermitilis fabC3 NP_(—)823467; S.avermitilisfabF NP_(—)823468; Bacillus subtilis fabH_A NP_(—)389015; B.subtilis fabHB NP_(—)388898; B. subtilis ACP NP_(—)389474; B. subtilisfabF NP_(—)389016; Stenotrophomonas maltophilia SmaIDRAFT_(—)0818ZP_(—)01643059; S. maltophilia SmaIDRAFT_(—)0821 ZP_(—)01643063; S.maltophilia SmaIDRAFT_(—)0822 ZP_(—)01643064; Legionella pneumophilafabHYP_(—)123672;L. pneumophila ACP YP_(—)123675; and L. pneumophilafabF YP_(—)123676.

Other branched alkane production genes which can be expressed include S.coelicolor ccr (EC 1.1.19, NP_(—)630556) crotonyl-CoA reductase, S.coelicolor icmA (EC 5.4.99.2, NP_(—)629554) isobuturyl-CoA mutase largesubunit, and S. coelicolor icmB (EC 5.4.99.13, NP_(—)630904)isobuturyl-CoA mutase small subunit; or Streptomyces cinnamonensis ccr(EC 1.1.19, AAD53915) crotonyl-CoA reductase, S. cinnamonensis icmA (EC5.4.99.2, AAC08713) isobuturyl-CoA mutase large subunit, and S.cinnamonensis icmB (EC 5.4.99.13, AJ246005) isobuturyl-CoA mutase smallsubunit.

Step 3 includes to the three genes above ccr, icmA and icmB expressingbranched alkanes, an alcohol reductase activity A. baylyi acrl expressedwith bkd operon, which yields isopentanol, isobutanol or 2-methylbutanol.

Similarly, expression of genes associated with the genes above, A.baylyi acrl alcohol reductase, express with ccr/icm genes pathwaysabove, yields isobutanol.

Example 12 Production of Fatty Acids with Genes in Host which MayInterfere with Branch FAS

To increase branched FAS, lower expression fabH (EC 2.3.1.41)β-ketoacyl-ACP synthase III or lower expression fabF (EC 2.3.1.41)β-ketoacyl-ACP synthase II.

Example 13 Production of ω-Cyclic Fatty Acids

Genes needed to be expressed which provide the cyclic precursorcyclohexylcarbonyl-CoA, and can be expressed with the branch-tolerantFAS genes include, for example, bkdC, lpd, fabH, ACP, fabF, fabH1, ACP,fabF, fabH3, fabC3, fabF, fabH_A, fabH_B, ACP.

Example 14 Ansatrienin Cluster

Express 2-cyclohexenylcarbonyl CoA isomerase Streptomyces collinus ansJK(AF268489), S. collinus ansL (AF268489), 1-cyclohexenylcarbonyl CoAreductase S. collinus chcA (U72144), and acyl CoA isomerase S. collinuschcB (AF268489).

Example 15 Phoslactomycin Cluster

The following genes are co-expressed with chcB from S. collinus, S.coelicolor or S. avermitilis: express 5-enolpyruvylshikimate-3-phosphatesynthase from Streptomyces sp. HK803 plmJK (AAQ84158), acyl-CoAdehydrogenase from Streptomyces sp. HK803 plmL (AAQ84159), enoyl-(ACP)reductase from Streptomyces sp. HK803 chcA (AAQ84160), 2,4-dienoyl-CoAreductase Streptomyces sp. HK803 plmM (AAQ84161), and acyl CoA isomerasefrom S. coelicolor chcB/caiD (NP_(—)629292).

Example 16 Fatty Acid/Alkane Export

Express alkane transporter such as Rhodococcus erythopolis ansP(AAN73268), cer5, ABC transporter such as A. thalania AtIg51500(AY734542), multi-drug efflux protein E. coli acrAB (NP_(—)414996.1,NP_(—)414995.1), efflux protein E. coli tolC(NP_(—)417507.2), multi-drugefflux protein E. coli AcrEF (NP_(—)417731.1, NP_(—)417732.1), T.elongatus BP-1 tll1618, (NP_(—)682408.1), T. elongatus BP-1 tll111619(NP_(—)682409.1), T. elongatus BP-1 tll0139 (NP_(—)680930.1), mammalianfatty acid transport protein (FATP) from D. melanogaster, C. elegans,fatty acid transport protein (FATP) from M. tuberculosis, mammalianfatty acid transport protein (FATP) from S. cerevisiae, transporterAcinetobacter sp. HO1-N.

Example 17 Biodiesel-Like Biosynthesis

A cyanobacterium strain is transformed with the plasmids carrying a waxsynthase gene from A. baylyi (EC: 2.3.175), a thioesterase gene fromCuphea hookeriana (EC AAC72883) and a fadD gene from E. coli. Thisrecombinant strain is grown at optimal temperatures under certainconditions in suitable media. The cells are separated from the spentmedium by centrifugation. The cell pellet is re-suspended and the cellsuspension and the spent medium are then extracted with ethyl acetate.The resulting ethyl acetate phases from the cells suspension and thesupernatant are subjected to GC-MS analysis. The fatty acid esters arequantified using commercial palmitic acid ethyl ester as the reference.Fatty acid esters are also made using the methods described hereinexcept that methanol, or isopropanol is added to the fermentation mediaand the expected fatty acid esters are produced.

Wax synthase (EC 2.3.1.75) generates acyl ester from acyl-CoA andterminal acyl-OH. Acetyl transferase (EC 2.3.1.84) converts alcohol andacetyl-CoA to acetic ester and CoA. The following are exemplary genes tobe expressed:

-   -   wst9 from Fundibacter jadensis DSM 12178    -   wshn from Acinetobacter sp. HO1-N    -   wsadp1 from Acinetobacter baylyi ADP 1    -   mWS from H. sapiens    -   m WS from Mus musculus (Q6E1M8)    -   SAAT from Fragaria xananassa    -   mpAAT from Malus xdomestica    -   JjWS from Simmondsia chinensis

Additional genes for the production of biodiesel are set forth in Table11, below:

TABLE 11 GenBank: Genbank: Producing organism Notes gene protein E. colicarboxy transferase, NC_000913.2 NP_414727 alpha subunit E. coli biotincarboxyl carrier NC_000913.2 NP_417721 protein (BCCP) E. coli biotincarboxylase NC_000913.2 NP_417722 E. coli caboxytransferase, NC_000913.2NP_416819 beta subunit Synechococcus sp. accA from thermophilicNC_007776 JA-2-3B′a(2-13) cyanobacterium Synechococcus sp. accB fromthermophilic NC_007776 JA-2-3B′a(2-13) cyanobacterium Synechococcus sp.accC from thermophilic NC_007776 JA-2-3B′a(2-13) cyanobacteriumSynechococcus sp. accD from thermophilic NC_007776 JA-2-3B′a(2-13)cyanobacterium Cuphea hookeriana C-8:0 to C-10:0 U39834.1 AAC49269thioesterase Umbellularia C-12:0 M94159.1 Q41635 california thioesteraseCinnamonum C-14:0 U17076.1 Q39473 camphorum thioesterase E. coli C-18:1NC_000913 NP_415027 thioesterase E. coli flexible NC_000913 NP_416319.1Synthetase Trichodesmium best blast match of 7002 NC_008312 YP_722779erythraeum IMS101 acyl-CoA Synthetase Synechococcus sp. putative fattyacid-CoA NC_007776 YP_478389.1 JA-2-3B′a(2-13) ligase from thermophileAcinetobacter acyl-CoA to aldehyde (host U77680.1 AAC45217 baylyi ADP1to OH) known range C14 and higher but possibly to C8-NADPH Synechococcussp. homology to acr1 from NC_007776 YP_476452.1 JA-2-3B′a(2-13)cyanobacterial thermophile Simmondsia acyl-CoA to fatty alcohol AF149917AAD38039.1″ chinensis via aldehyde (2 NADPH) native range C20 to C22,can handle C16-C18 Rubrobacter bacterial analog of jojoba NC_008148.1YP_644868 xylanophilus FAR from thermophile DSM 9941 Acinetobacterthermostable fatty aldehyde AB047854 BAB12270.1 sp. M-1 reductase,C2-C14 (NADPH) Arabidopsis Note: plant transmembrane NM_100101.3NP_171723 thaliana protein, C18-C32 Thermosynechococcus steroldesaturase family NP_682707 elongatus BP-1 protein; E(cer1) = 1e−07Parvularcula COG3000 Sterol desaturase, NZ_AAMU01000002.1 ZP_01017596bermudensis E(cer1) = 1e−08 HTCC2503 Pedobacter sp. sterol desaturasefamily NZ_ABCM01000018.1 ZP_01885831 BAL39 protein, E(cer1) = 1e−08Fragaria x ananassa Note: modified to remove AF193789 AAG13130.1 NcoIsite for E. coli exp. Functioned as wax synthase, not acetyl transferaseAcinetobacter can make smaller waxes like AF529086.1 AAO17391.1 baylyiADP1 octyl octanoate as can SAAT

Genes and Plasmids: The E. coli thioesterase tesA gene with the leadersequence targeting the removed (Genbank #NC_(—)000913, ref: Chot andCronan, 1993), the E. coli acyl-CoA synthetase fadD (Genbank#NC_(—)000913, ref: Kameda and Nunn, 1981) and the wax synthase (=wax)from Acinetobacter baylyi strain ADPI (Genbank #AF529086. 1, ref:Stöveken et al. 2005) was purchased from DNA 2.0 following codonoptimization, checking for secondary structure effects, and removal ofany unwanted restriction sites (NdeI, XhoI, BamHI, NgoMIV, NcoI, SacI,BsrGI, AvrII, BmtI, MluI, EcoRI, SbfI, NotI, SpeI, XbaI, PacI, AscI,FseI). These genes were received on pJ201 vectors and assembled into athree gene operon (tesA-fadD-wax, SEQ ID NO: 3) with flanking NdeI-EcoRIsites on the recombination vector pJB5 under the control of the PaphIIkanamycin resistance cassette promoter. Another plasmid was constructedwhere the PaphII promoter was replaced with a Ptrc promoter under thecontrol of a lacIq repressor (SEQ ID NO: 4). A control plasmid with onlytesA under the control of the PaphII promoter was also prepared. TheJoule plasmid numbers for these three plasmids are pJB494, pJB532, andpJB413, respectively.

The pJB5 base vector was designed as an empty expression vector forrecombination into Synechococcus sp. PCC 7002. Two regions of homology,the Upstream Homology Region (UHR) and the Downstream Homology Region(DHR) were designed to flank the construct. These 500 bp regions ofhomology correspond to positions 3301-3800 and 3801-4300 (GenbankAccession NC_(—)005025) for UHR and DHR respectively. The aadA promoter,gene sequence, and terminator were designed to confer spectinomycin andstreptomycin resistance to the integrated construct. For expression,pJB5 was designed with the aph2 kanamycin resistance cassette promoterand ribosome binding site (RBS). Downstream of this promoter and RBS, wedesigned and inserted the restriction endonuclease recognition site forNdeI and EcoRI, as well as the sites for SpeI and PacI. Following theEcoRI site, the natural terminator from the alcohol dehydrogenase genefrom Zymomonas mobilis (adhII) terminator was included. Convenient xbaIrestriction sites flank the UHR and the DHR allowing cleavage of the DNAintended for recombination from the rest of the vector. The pJB5 vectorwas constructed by contract synthesis from DNA2.0 (Menlo Park, Calif.).

Strain Construction: The constructs as described above were integratedonto the plasmid pAQ1 in Synechococcus sp. PCC 7002 using the followingprotocol. Synechococcus 7002 was grown for 48 h from colonies in anincubated shaker flask at 37° C. at 2% CO₂ to an OD₇₃₀ of 11n A⁺ mediumdescribed in Frigaard et al., Methods Mol. Biol., 274:325-340 (2004).450 μL of culture was added to a epi-tube with 50 μL of 5 μg of plasmidDNA digested with xbaI ((New England Biolabs; Ipswitch, Mass.)) that wasnot purified following restriction digest. Cells were incubated in thedark for four hours at 37° C. The entire volume cells was plated on A⁺medium plates with 1.5% agarose and grown at 37° C. in a lightedincubator (40-60 μE/m2/s PAR, measured with a LI-250A light meter(LI-COR)) for about 24 hours. 25 μg/mL of spectinomycin was underlayedon the plates. Resistant colonies were visible in 7-10 days afterfurther incubation, and recombinant strains were confirmed by PCR usinginternal and external primers to check insertion and confirm location ofthe genes on pAQ1 in the strains (Table 12).

TABLE 12 Joule Culture Collection (JCC) numbers of the Synechococcus sp.PCC 7002 recombinant strains with gene insertions on the native plasmidpAQ1 JCC # Promoter Genes Marker JCC879 PaphII — aadA JCC750 PaphII tesAaadA JCC723 PaphII tesA-fadD-wax aadA JCC803 lacIq Ptrc tesA-fadD-waxaadA

Ethyl Ester Production culturing conditions: One colony of each of thefour strains (Table 12) was inoculated into 10 mls of A+ mediacontaining 50 μg/ml spectinomycin and 1% ethanol (v/v). These cultureswere incubated for about 4 days in a bubble tube at 37° C. sparged atapproximately 1-2 bubbles of 1% CO₂/air every 2 seconds in light (40-50μE/m2/s PAR, measured with a LI-250A light meter (LI-COR)). The cultureswere then diluted so that the following day they would have OD₇₀₃ of2-6. The cells were washed with 2×10 ml JB 2.1/spec200, and inoculatedinto duplicate 28 ml cultures in JB 2.1/spec200+1% ethanol (v/v) mediato an OD₇₃₀=0.07. IPTG was added to the JCC803 cultures to a finalconcentration of 0.5 mM. These cultures were incubated in a shakingincubator at 150 rpm at 37° C. under 2% CO₂/air and continuous light(70-130 μE m2/s PAR, measured with a LI-250A light meter (LI-COR)) forten days. Water loss through evaporation was replaced with the additionof sterile Milli-Q water. 0.5% (v/v) ethanol was added to the culturesto replace loss due to evaporation every 48 hours. At 68 and 236 hours,5 ml and 3 ml of culture were removed from each flask for ethyl esteranalysis, respectively. The OD₇₃₀s reached by the cultures is given inTable 13.

TABLE 13 The OD₇₃₀s reached by the Synechococcus sp. PCC 7002recombinant strains at timepoints 68 and 236 h JCC879 JCC879 JCC750JCC750 JCC723 JCC723 JCC803 JCC803 Time point #1 #2 #1 #2 #1 #2 #1 #2 68 h 3.6 4.0 4.6 5.0 6.6 6.0 5.4 5.8 236 h 21.2 18.5 19.4 20.9 22.221.4 17.2 17.7

The culture aliquots were pelleted using a Sorvall RC6 Plus superspeedcentrifuge (Thermo Electron Corp) and a F13S-14X50CY rotor (5000 rpm for10 min). The spend media supernatant was removed and the cells wereresuspended in 1 ml of Milli-Q water. The cells were pelleted againusing a benchtop centrifuge, the supernatant discarded and the cellpellet was stored at −80° C. until analyzed for the presence of ethylesters.

Detection and quantification of ethyl esters in strains: Cell pelletswere thawed and 1 ml aliquots of acetone (Acros Organics 326570010)containing 100 mg/L butylated hydroxytoluene (Sigma-Aldrich B1378) and50 mg/L ethyl valerate (Fluka 30784) were added. The cell pellets weremixed with the acetone using a Pasteur pipettes and vortexed twice for10 seconds (total extraction time of 1-2 min). The suspensions werecentrifuged for 5 min to pellet debris, and the supernatants wereremoved with Pasteur pipettes and subjected to analysis with a gaschromatograph using flame ionization detection (GC/FID).

An Agilent 7890A GC/FID equipped with a 7683 series autosampler was usedto detect the ethyl esters. One μL of each sample was injected into theGC inlet (split 5:1, pressure: 20 psi, pulse time: 0.3 min, purge time:0.2 min, purge flow: 15 mL/min) and an inlet temperature of 280° C. Thecolumn was a HP-5MS (Agilent, 30 m×0.25 mm×0.25 μm) and the carrier gaswas helium at a flow of 1.0 mL/min. The GC oven temperature program was50° C., hold one minute; 10°/min increase to 280° C.; hold ten minutes.The GC/MS interface was 290° C., and the MS range monitored was 25 to600 amu. Ethyl myristate [retention time (rt): 17.8 min], ethylpalmitate (rt: 19.8 min) and ethyl stearate (rt: 21.6 min) wereidentified based on comparison to a standard mix of C4-C24 even carbonsaturated fatty acid ethyl esters (Supelco 49454-U). Ethyl oleate (rt:21.4 min) was identified by comparison with an ethyl oleate standard(Sigma Aldrich 268011). These identifications were confirmed by GC/MS(see following Methyl Ester Production description for details).Calibration curves were constructed for these ethyl esters using thecommercially available standards, and the concentrations of ethyl esterspresent in the extracts were determined and normalized to theconcentration of ethyl valerate (internal standard).

Four different ethyl esters were found in the extracts of JCC723 andJCC803 (Table 14). In general, JCC803 produced 2-10× the amount of eachethyl ester than JCC723, but ethyl myristate was only produced in lowquantities of 1 mg/L or less for all these cultures. No ethyl esterswere found in the extracts of JCC879 or JCC750, indicating that thestrain cannot make ethyl esters naturally and expression of only tesA isnot enough to confer production of ethyl esters (FIG. 20).

TABLE 14 Amounts of respective ethyl esters found in the cell pelletextracts of JCC723 given as mg/L of culture C14:0 C16:0 C18:1 C18:0ethyl ethyl ethyl ethyl Sample ester ester ester ester JCC723 #1 68 h0.08 0.34 0.22 0.21 JCC723 #2 68 h 0.12 1.0 0.43 0.40 JCC803 #1 68 h0.45 6.6 1.4 0.74 JCC803 #2 68 h 0.63 8.6 2.0 0.94 JCC723 #1 236 h 1.0415.3 2.1 4.5 JCC723 #2 236 h 0.59 9.0 1.3 3.7 JCC803 #1 236 h 0.28 35.313.4 19.2 JCC803 #2 236 h 0.49 49.4 14.9 21.2

Methyl Ester Production Culturing conditions: One colony of JCC803(Table 1) was inoculated into 10 mls of A+ media containing 50 μg/mlspectinomycin and 1% ethanol (v/v). This culture was incubated for 3days in a bubble tube at 37° C. sparged at approximately 1-2 bubbles of1% CO₂/air every 2 seconds in light (40-50 μE/m2/s PAR, measured with aLI-250A light meter (LI-COR)). The culture was innoculated into twoflasks to a final volume of 20.5 ml and OD₇₃₀=0.08 in A+ mediacontaining 200 μg/ml spectinomycin and 0.5 mM IPTG with either 0.5%methanol or 0.5% ethanol (v/v). These cultures were incubated in ashaking incubator at 150 rpm at 37° C. under 2% CO₂/air and continuouslight (70-130 μE m2/s PAR, measured with a LI-250A light meter (LI-COR))for three days. Water loss through evaporation was replaced with theaddition of sterile Milli-Q water. 5 ml of these cultures (OD₇₃₀=5-6)were analyzed for the presence of ethyl or methyl esters.

Detection of ethyl- or methyl-esters: Cell pellets were thawed and 1 mlaliquots of acetone (Acros Organics 326570010) containing 100 mg/Lbutylated hydroxytoluene (Sigma-Aldrich B1378) and 50 mg/L ethylvalerate (Fluka 30784) were added. The cell pellets were mixed with theacetone using a Pasteur pipettes and vortexed twice for 10 seconds(total extraction time of 1-2 min). The suspensions were centrifuged for5 min to pellet debris, and the supernatants were removed with Pasteurpipettes and subjected to analysis with a gas chromatograph using massspectral detection (GC/MS).

An Agilent 7890A GC/5975C E1-MS equipped with a 7683 series autosamplerwas used to measure the ethyl esters. One μL of each sample was injectedinto the GC inlet using pulsed splitless injection (pressure: 20 psi,pulse time: 0.3 min, purge time: 0.2 min, purge flow: 15 mL/min) and aninlet temperature of 280° C. The column was a HP-5MS (Agilent, 30 m×0.25mm×0.25 μm) and the carrier gas was helium at a flow of 1.0 mL/min. TheGC oven temperature program was 50° C., hold one minute; 10°/minincrease to 280° C.; hold ten minutes. The GC/MS interface was 290° C.,and the MS range monitored was 25 to 600 amu. Compounds indicated bypeaks present in total ion chromatograms were identified by matchingexperimentally determined mass spectra associated with the peaks withmass spectral matches found by searching in a NIST 08 MS database.

The culture of JCC803 incubated with ethanol contained ethyl palmitate[retention time (rt): 18.5 min], ethyl heptadecanoate (rt: 19.4 min),ethyl oleate (rt: 20.1 min) and ethyl stearate (rt: 20.3 min). No ethylesters were detected in the strain incubated with methanol. Instead,methyl palmitate (rt: 17.8 min), methyl heptadecanoate (rt: 18.8 min)and methyl stearate were found (FIG. 21). This strain apparently has thecapability to make both methyl and ethyl esters depending on alcoholused. The wax synthase gene used in this strain is known to have a verybroad substrate specificity (Stöveken et al. 2005; Kalscheuer et al.2006a; Kalscheuer et al. 2006b), and therefore JCC803 could utilize awide variety of alcohols to produce various fatty acid esters.

REFERENCES

-   Cho, H. and Cronan, J. E. 1993. Escherichia coli thioesterase I,    molecular cloning and sequencing of the structural gene and    identification as a periplasmic enzyme. The Journal of Biological    Chemistry 268: 9238-9245.-   Kalscheuer, R., Stölting, T. and Steinbüchel, A. 2006a. Microdiesel:    Escherichia coli engineered for fuel production. Microbiology 152:    2529-2536.-   Kalscheuer, R., Stöveken, T., Luftman, H., Malkus, U., Reichelt, R.    and Steinbüchel, A. 2006b. Neutral lipid biosynthesis in engineered    Escherichia coli: jajoba oil-like wax esters and fatty acid butyl    esters. Applied and Environmental Microbiology 72: 1373-1379.-   Kameda, K. and Nunn, W. D. 1981. Purification and characterization    of the acyl Coenzyme A synthetase from Escherichia coli. The Journal    of Biological Chemistry 256: 5702-5707.-   Stöveken, T., Kalscheuer, R., Malkus, U., Reichelt, R. and    Steinbüchel, A. 2005. The wax ester synthase/acyl coenzyme A:    diacylglycerol acyltransferase from Acinetobacter sp. strain ADP 1:    characterization of a novel type of acyltransferase. Journal of    Bacteriology 187:1369-1376.

Example 18 Fatty Alcohol Producers

Acyl-CoA reductases (EC 1.2.1.50) convert acyl-CoA and NADPH to fattyalcohol and CoA. Examples of genes to express include: bfar from Bombyxmori (Q8R079); acrl from Acinetobacter baylyi ADP1 (AAC45217); jjfarfrom Simmondsia chinensis; an unspecified acyl-CoA reductase fromTriticum aestivum; mfar1 from Mus musculus; mfar2 from Mus musculus;acrM1 from Acinetobacter sp. M1; and hfar from H. sapiens.

Example 19 Engineered Microorganisms Producing Octane

To produce a particular alkane such as octane, several genes asidentified in FIG. 1 are introduced in a selected microorganism. Theenzyme acetyl-CoA:ACP transacylase (E. coli fabH) (EC 2.3.1.38)generates acetyl-ACP+CoA from acetyl-CoA and ACP. Acetyl-CoA carboxylase(E. coli accBCAD) (EC 6.4.1.2) generates malonyl-CoA from acetyl-CoA,ATP and CO2. Malonyl-CoA:ACP transacylase (E. coli fabD) (EC 2.3.1.39)generates malonyl-ACP and CoA from malonyl-CoA and ACP. 3-ketoacyl-ACPsynthase (E. coli fabB) (EC 2.3.1.41) generates CO2 and 3-ketoacyl-ACPfrom acyl-ACP and malonyl-ACP. 3-Ketoacyl-ACP reductase (E. coli fabG)(EC 1.1.1.100) generates 3-hydroxyacyl-ACP from 3-ketoacyl-ACP andNADPH. 3-hydroxyacyl-ACP dehydratase (E. coli fabA) (EC 4.2.1.60)generates enoyl-ACP from 3-hydroxyacyl-ACP. Enoyl-ACP reductase (E. colifab1) (EC 1.3.1. {9,10}) generates acyl-ACP from enoyl-ACP and NADH,NADPH. Acyl-ACP hydrolase (S. cerevisiae fas1) (EC 3.1.2.14) generatesfatty acid and ACP from acyl-ACP. Several aldehyde dehydrogenase foundin P. aeruginosa (EC 1.2.1. {3,4}) generate octanal from octanoate andNADH, NADPH. Alcohol dehydrogenase (Z. mobilis adh1) (EC 1.1.1. {1,2})generates 1-octanol from octanal and NADH, NADPH. Alkane 1-monooxygenase(P. fluorescens alkB) (EC 1.14.15.3) then generates n-octane, NAD(P)Hand O2 from 1-octanol. Production of n-octane confers engineered hostcell expression of the above enzyme activities.

Example 20 Production of Isoprenoids

To generate a strain of cyanobacteria for the production of3-methyl-but-3-en-1-ol and 3-methyl-but-2-en-1-ol, plasmids aregenerated by inserting a genomic DNA fragment of Synnechococcus sp. PCC7002 comprising the coding sequence of the nudF gene and upstreamgenomic sequences into a vector.

Synechococcus 7002 is grown for 48 h from colonies in an incubatedshaker flask at 30° C. at 1% CO₂ to an OD₇₃₀ of 11n A⁺ medium describedin Frigaard et al., Methods Mol. Biol. 274:325-340 (2004). 500 μL ofculture is added to a test-tube with 30 μL of 1-5 μg of DNA prepped froma Qiagen Qiaprep Spin Miniprep Kit (Valencia, Calif.) for eachconstruct. Cells were incubated bubbling in 1% CO₂ at approximately 1bubble every 2 seconds for 4 hours. 200 μL of cells were plated on A⁺medium plates with 1.5% agarose and grown at 30° C. for two days in lowlight. 10 μg/mL of spectinomycin was underplayed on the plates.Resistant colonies were visible in 7-10 days. The cultures are grownovernight by shaking on a rotary shaker. The OD₆₀₀ of each culture ismeasured, and a sample is removed. To each removed sample, ethyl acetateis added, and the sample is vortexed. A portion of the upper ethylacetate phase is transferred to a clean glass vial for analysis by gaschromatography-mass spectrometry.

The samples are analyzed on a GC/MS. A 1 μL sample is separated on theGC using a DB-5 column (Agilent Technologies, Inc., Palo Alto, Calif.)and helium carrier gas. The oven cycle for each sample is 60° C. for 3minutes, increasing temperature at 60° C./minute to a temperature of300° C., and a hold at 300° C. for 2 minutes. The total run time is 9minutes. The resolved samples are analyzed by a mass-selective detectoralong with previously measured retention time of 3-methyl-3-buten-1-oland 3-methyl-2-buten-1-ol mass spectra using this GC protocol.

The 3-methyl-3-buten-1-ol and isoamyl alcohol can be blendedrespectively with a California Reformulated Gasoline Blendstock forOxygen Blending (CARBOB) to form various mixtures having an oxygencontent of 2 wt %, 2.7 wt. % or 3.5 wt. %. Similarly, 1-butanol,ethanol, methyl tertiary-butyl ether (MTBE) and ethyl tertiary-butylether (ETBE) can also be blended respectively with CARBOB to formvarious mixtures having an oxygen content of 2 wt %, 2.7 wt. % or 3.5wt. %. The API gravity values, research octane numbers, motor octanenumbers, anti-knock indexes, vapor pressure data, net heats ofcombustion, water tolerance data, and vapor-liquid ratio of the mixturesare tested.

Example 21 Engineered Microorganisms Producing Terephthalate

2-dehydro-3-deoxyphosphoheptonate aldolase E. coli aroF (EC 2.5.1.54)generates 3-deoxy-D-arabino-heptulosonate-7-P from PEP andD-erythrose-4-P. 3-dehydroquinate synthase E. coli aroB (EC 4.2.3.4)generates 3-dehydroquinate from 3-deoxy-D-arabino-heptulosonate-7-P.3-dehydroquinate dehydratase E. coli aroD (EC 4.2.1.10) generates3-dehydro-shikimate from 3-dehydroquinate. 3-dehydroshikimatedehydratase from Acinetobacter sp. ADP1 quiC (EC 4.2.1.n) generatesprotocatechuate from 3-dehydro-shikimate. β-ketoadipyl-CoA synthase fromRhodococcus sp. RHA1 pcaF (EC 2.3.1.174) generates β-ketoadipyl-CoA andCoA from acetyl-CoA and succinyl-CoA. β-ketoadipate CoA-transferase fromPseudomonas putida pcalJ (EC 2.8.3.6) generates β-ketoadipate andsuccinyl-CoA from β-ketoadipyl-CoA and succinate. 3-oxoadipateenol-lactone hydrolase Rhodococcus sp. RHA1 pcaL EC 3.1.1.24 generatesβ-ketoadipate enol lactone from β-ketoadipate. 4-carboxymuconolactonedecarboxylase Rhodococcus sp. RHA1 pcaL (EC 4.1.1.44) generatesγ-carboxy-muconolactone from β-ketoadipate enol lactone and CO2.γ-carboxy-cis,cis-muconate cycloisomerase Rhodococcus sp. RHA1 pcaB (EC5.5.1.2) generates β-carboxy-cis,cis-muconate fromγ-carboxy-muconolactone. Protocatechuate 3,4-dioxygenase fromRhodococcus sp. RHA1 pcaGH(EC 1.13.11.3) generates protocatechuate fromβ-carboxy-cis,cis-muconate. Protocatechuate 1,2-cis-dihydrodioldehydrogenase Rhodococcus sp. RHA1 tpaC (EC 1.3.1.n) generates DDT fromprotocatechuate, CO₂ and NADPH. Terephthalate 1,2-dioxygenaseRhodococcus sp. RHA1 tpaAB (EC 1.14.12.15) converts DDT toterephthalate, NADH and O2.

Example 22 Engineered Microorganisms Producing 1,3-Propanediol

The enzyme sn-glycerol-3-P dehydrogenase S. cerevisiae dar1 (EC 1.1.1.{8,94}) generates sn-glycerol-3-P from dihydroxyacetone-P and {NADH,NADPH}. sn-glycerol-3-phosphatase S. cerevisiae gpp2 (EC 3.1.3.21)generates glycerol from sn-glycerol-3-P. glycerol dehydratase K.pneumonia dhaB1-3 (EC 4.2.1.30) generates 3-hydroxypropanal fromglycerol. 1,3-propanediol oxidoreductase K. pneumonia dhaT (EC1.1.1.202) converts 3-hydroxypropanal and NADH to 1,3-propanediol.

Example 23 Engineered Microorganisms Producing 1,4-butanediol

Succinyl-CoA dehydrogenaseC. Kluyveri suc D (EC 1.2.1.n) generatessuccinic from succinyl-CoA and NADPH. 4-hydroxybutyrate dehydrogenase A.thaliana ghbdh (EC 1.1.1.2) generates 4-hydroxybutyrate from succinicsemialdehyde and NADPH. Glutamate dehydrogenase E. coli gdhA (EC1.4.1.4) generates glutamate from α-ketoglutarate, NH3 and NADPH.Glutamate decarboxylase E. coli gadA (EC 4.1.1.15) generates4-aminobutyrate and CO2 from glutamate. 4-aminobutyrate transaminase E.coli gabT (EC 2.6.1.19) generates glutamate and succinic semialdehydefrom 4-aminobutyrate and α-ketoglutarate. Aldehyde dehydrogenase E. colialdH (EC 1.1.1.n) generates 4-hydroxybutanal from 4-hydroxybutyrate andNADH. 1,3-propanediol oxidoreductase K. pneumonia dhaT (EC 1.1.1.202)generates 1,4-butanediol from 4-hydroxybutanal and NADH. This scheme isillustrated in FIG. 8. FIG. 8 also illustrates genes that can be knockedout (if already present in a host cell) to improve efficiency of1,4-butanediol synthesis. Those genes are identified by the “Xs.” Genesto be overexpressed are indicated in FIG. 8 by the colored arrows.

Example 24 Engineered Microorganisms Producing PHB

Beta-ketothiolase (R. eutropha phaA) (EC 2.3.1.16) converts 2 acetyl-CoAto acetoacetyl-CoA and CoA. Acetoacetyl-CoA reductase (R. eutropha phaB)(EC 1.1.1.36) generates 3-hydroxybutyryl-CoA from acetoacetyl-CoA andNADPH. PHA synthase (R. eutropha phaC) generates PHB and CoA from3-hydroxybutyryl-CoA.

Production of Polyhydroxybutyrate (PHB): PHB is produced in nature bymicroorganisms as a carbon reservoir, usually under conditions of carbonsufficiency and limitation of some other nutrient. PHB is synthesizedinside the cell in all known cases, not secreted into the medium. PHB isderived from acetyl-CoA in a three-step enzymatic pathway consisting ofacetyl-CoA:acetyl-CoA C-acetyltransferase (EC 2.3.1.9), which convertstwo molecules of acetyl-CoA to one molecule of acetoacetyl-CoA and onemolecule of free CoA; (R)-3-hydroxyacyl-CoA:NADP+ oxidoreductase (EC1.1.1.36), which reduces acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA atthe expense of NADPH; and polyhydroxyalkanoate synthase (EC 2.3.1.-),which polymerizes units of (R)-3-hydroxybutyryl-CoA by adding each tothe growing chain and liberating free CoA.

Microorganisms that naturally produce PHB also have the capacity todegrade it, via one or more poly[(R)-3-hydroxybutanoate]hydrolase (EC3.1.1.75) enzymes, more commonly referred to as depolymerases. Theseenzymes are expressed or activated to access stored carbon and energyupon occurrence of the appropriate conditions, such as carbonlimitation. Expression of the PHB pathway in a non-natural produceroften leads to irreversible accumulation of PHB because the non-naturalproducer lacks depolymerase activity.

Methods of Detecting PHB: Intracellular PHB can be measured by solventextraction and esterification of the polymer from whole cells.Typically, lyophilized biomass is extracted with methanol-chloroformwith 10% HCl as a catalyst. The chloroform dissolves the polymer, andthe methanol esterifies it in the presence of HCl. The resulting mixtureis extracted with water to remove hydrophilic substances, and theorganic phase is analyzed by GC.

Engineered Microorganisms Producing PHB: The phaCAB operon fromRalstonia eutropha H16 is expressed in the recombinant host undercontrol of an appropriate promoter. Examples of such promoters includethe aphII, cpcB, cI, and lacIq-trc promoters. The operon is placed onpAQ1, pAQ7, or at a suitable site on the chromosome by homologousrecombination.

Construction of pJB528: The DNA sequence encoding the PHB operon ofRalstonia eutropha H16 (phaCAB) was obtained from GenBank (NC008313,Ralstonia eutropha H16 chromosome 1). The individual genes (gene locustags H16-A1437, H16-A1438, and H16-A1439) were each codon-optimized forexpression in E. coli. The genes were then recast into an operon in theform phaCAB, but with convenient restriction sites between the genes.This optimized phaCAB operon was obtained by contract synthesis from DNA2.0 (Menlo Park, Calif.). The phaCAB operon was designed with an NdeIsite including part of the start codon and an EcoRI site after the stopcodon. This construct was removed from its backbone vector byrestriction digest with NdeI and EcoRI and inserted by ligation intopAQ1 insertion vector pJB496 (SEQ ID NO: 5) that had been digested withthe same enzymes. The ligated construct, pJB528, was transformed into E.coli CopyCutter (Epicentre; Madison, Wis.). Subsequent transformationinto E. coli NEB5c (New England Biolabs; Ipswich, Mass.) gaveintracellular PHB granules which were apparent by visual inspection withlight microscopy.

Example 25 Engineered Microorganisms Producing Acrylate

Enoyl-CoA hydratase (E. coli paaF) (EC 4.2.1.17) converts3-hydroxypropionyl-CoA to acryloyl-CoA. Acrylate CoA-transferase (R.eutropha pct) (EC 2.8.3.n) generates acrylate+acetyl-CoA fromacryloyl-CoA and acetate.

Example 26 Engineered Microorganisms Producing ε-Caprolactone

Acetyl-CoA:ACP transacylase E. coli fabH (EC 2.3.1.38) generatesacetyl-ACP and CoA from acetyl-CoA and ACP. Acetyl-CoA carboxylase E.coli accBCAD (EC 6.4.1.2) generates malonyl-CoA acetyl-CoA, ATP and CO₂.Malonyl-CoA:ACP transacylase E. coli fabD (EC 2.3.1.39) generatesmalonyl-ACP and CoA from malonyl-CoA and ACP. 3-ketoacyl-ACP synthase E.coli fabB (EC 2.3.1.41) generates CO2 and 3-ketoacyl-ACP from acyl-ACPand malonyl-ACP. 3-ketoacyl-ACP reductase E. coli fabG (EC 1.1.1.100)generates 3-hydroxyacyl-ACP from 3-ketoacyl-ACP and NADPH.3-hydroxyacyl-ACP dehydratase E. coli fabA (EC 4.2.1.60) generatesenoyl-ACP from 3-hydroxyacyl-ACP. Enoyl-ACP reductase E. coli fab1 (EC1.3.1. {9,10}) generates acyl-ACP from enoyl-ACP and {NADH, NADPH}.Acyl-ACP hydrolase S. cerevisiae FAS1 (EC 3.1.2.14) generates fatty acidand ACP from acyl-ACP. Fatty-acid monooxygenase P. oleovorans alkB (EC1.14.15.3) generates ω-hydroxyalkanoate from fatty acid, NADPH and O2.An 1,6-lactonase (EC 3.1.1.n) converts 6-hydroxyhexanoate toε-caprolactone.

Example 27 Engineered Microorganisms Producing Isoprene

1-deoxy-D-xylulose-5-phosphate synthase E. coli dxs (EC 2.2.1.7)generates 1-deoxy-D-xylulose-5-P and CO₂ from pyruvate andD-glyceraldehyde-3-P. 1-deoxy-D-xylulose-5-phosphate reductoisomerase E.coli dxr (EC 1.1.1.267) generates 2-C-methyl-D-erythritol-4-P from1-deoxy-D-xylulose-5-P+NADPH. 2-C-methyl-D-erythritol 4-phosphatecytidylyltransferase E. coli ispD (EC 2.7.7.60) generates4-(cytidine-5′-PP)-2-C-methyl-D-erythritol fromCTP+2-C-methyl-D-erythritol 4-P. 4-(cytidine5′-diphospho)-2-C-methyl-D-erythritol kinase E. coli ispE (EC 2.7.1.148)generates 2-P-4-(cytidine 5′-PP)-2-C-methyl-D-erythritol from ATP and4-(cytidine-5′-PP)-2-C-methyl-D-erythritol. 2-C-methyl-D-erythritol2,4-cyclodiphosphate synthase E. coli ispF (EC 4.6.1.12) generates2-C-methyl-D-erythritol-2,4-cyclo-PP+CMP from 2-P-4-(cytidine5′-PP)-2-C-methyl-D-erythritol. 4-hydroxy-3-methylbut-2-en-1-yldiphosphate synthase E. coli ispG (EC 1.17.4.3) generates(E)-4-hydroxy-3-methylbut-2-en-1-yl-PP from2-C-methyl-D-erythritol-2,4-cyclo-PP. 4-hydroxy-3-methylbut-2-enyldiphosphate reductase E. coli ispH (EC 1.17.1.2) generatesisopentenyl-PP and NADP from (E)-4-hydroxy-3-methylbut-2-en-1-yl-PP andNADPH. 4-hydroxy-3-methylbut-2-enyl diphosphate reductase E. coli ispH(EC 1.17.1.2) generates dimethylallyl-PP+NADP from(E)-4-hydroxy-3-methylbut-2-en-1-yl-PP and NADPH.Isopentenyl-diphosphate Δ-isomerase E. coli idi (EC 5.3.3.2) convertsdimethylallyl-PP to isopentenyl-PP.

Example 28 Engineered Microorganisms Producing Rubber

Rubber is produced by cis-polyprenylcistransferase from H. brasiliensis(EC 2.5.1.20), which converts isopentenyl-PP to rubber.

Example 29 Engineered Microorganisms Producing Lactate

Lactate dehydrogenase E. coli IdhA (EC 1.1.1.28) converts NADH andpyruvate to D-lactate.

Example 30 Engineered Microorganisms Producing DHA

DHA kinase C. freundii dhaK (EC 2.7.1.29) converts dihydroxyacetone andATP to dihydroxyacetone-P.

Example 31 Engineered Microorganisms Producing 3-hydroxypropionate

Acetyl-CoA carboxylase E. coli accBCAD (EC 6.4.1.2) generatesmalonyl-CoA from acetyl-CoA, ATP and CO2 (see, e.g., the pathway shownin FIG. 5). The bifunctional malonyl-CoA reductase from C. aurantiacus(EC 1.2.1.18, 1.1.1.59) converts malonyl-CoA and 2 NADPH to3-hydroxypropionate and CoA.

Example 32 Engineered Microorganisms Producing γ-Valerolactone

The enzyme 2-oxobutyrate synthase from C. pasteurianum (EC 1.2.7.2)converts propionyl-CoA, CO2 and 2 Fdred to 2-oxobutanoate, CoA and 2FDox. The enzyme 2-ethylmalate synthase from S. cerevisiae (EC 2.3.3.6)generates (R)-2-ethylmalate+CoA from 2-oxobutanoate and acetyl-CoA.Aconitase analog generates 3-carboxy-4-hydroxypentanoate from(R)-2-ethylmalate. Isocitrate dehydrogenase analog converts3-carboxy-4-hydroxypentanoate to levulinate. Acetoacetyl-CoA reductaseanalog R. eutropha ler generates 4-hydroxypentanoate from levulinate andNAD(P)H. 1,4-lactonase from R. norvegicus (EC 3.1.1.25) generatesγ-valerolactone from 4-hydroxypentanoate.

Alternatively, acetyl-CoA carboxylase E. coli accBCAD (EC 6.4.1.2)converts acetyl-CoA, ATP and CO2 to malonyl-CoA. The bifunctionalmalonyl-CoA reductase from C. aurantiacus (EC 1.2.1.18, 1.1.1.59)converts malonyl-CoA and 2 NADPH to 3-hydroxypropionate and CoA. Theenoyl-CoA hydratase E. coli paaF (EC 4.2.1.17) generates acryloyl-CoAfrom 3-hydroxypropionyl-CoA. Acyl-CoA dehydrogenase A. thalianaAt3G06810 (EC 1.3.99.3) generates propionyl-CoA from acryloyl-CoA andFADH2. Beta-ketothiolase R. eutropha bktB (EC 2.3.1.16) generates3-ketovaleryl-CoA and CoA from propionyl-CoA and acetyl-CoA.Acetoacetyl-CoA reductase R. eutropha phaB (EC 1.1.1.36) generates(R)-3-hydroxyvaleryl-CoA from 3-ketovaleryl-CoA and NADPH.3-hydroxybutyryl-CoA dehydratase X. axonopodis crt (EC 4.2.1.55)generates 3-pentenoyl-CoA from (R)-3-hydroxyvaleryl-CoA. Vinylacetyl-CoAΔ-isomerase C. dificile abfD (EC 5.3.3.3) generates4-hydroxypentanoyl-CoA from 3-pentenoyl-CoA. 4-hydroxybutyryl-CoAtransferase C. kluyveri or fZ (EC 2.8.3.n) converts4-hydroxypentanoyl-CoA and acetate to acetyl-CoA and4-hydroxypentanoate. 1,4-lactonase from R. norvegicus (EC 3.1.1.25)generates γ-valerolactone from 4-hydroxypentanoate.

Example 33 Engineered Microorganisms Producing Lysine

Aspartate aminotransferase E. coli aspC (EC 2.6.1.1) generatesL-aspartate and α-ketoglutarate from oxaloacetate and L-glutamate.Aspartate kinase E. coli lysC (EC 2.3.3.14) generates L-aspartyl-4-Pfrom L-aspartate and ATP. Aspartate semialdehyde dehydrogenase E. coliasd (EC 1.2.1.11) generates NADPH+L-aspartyl-4-phosphate fromL-aspartate-semialdehyde. Dihydrodipicolinate synthase E. coli dapA (EC4.2.1.52) generates L-2,3-dihydrodipicolinate from pyruvate andL-aspartate-semialdehyde. Dihydrodipicolinate reductase E. coli dapB (EC1.3.1.26) generates tetrahydrodipicolinate fromL-2,3-dihydrodipicolinate and NADPH. Tetrahydrodipicolinate succinylaseE. coli dapD (EC 2.3.1.117) generates N-succinyl-2-amino-6-ketopimelateand CoA from tetrahydrodipicolinate and succinyl-CoA.N-succinyldiaminopimelate-aminotransferase E. coli argD (EC 2.6.1.17)generates α-ketoglutarate and N-succinyl-L,L-2,6-diaminopimelate fromL-glutamate and N-succinyl-2-amino-6-ketopimelate.N-succinyl-L-diaminopimelate desuccinylase E. coli dapE (EC 3.5.1.18)generates L,L-diaminopimelate and succinate fromN-succinyl-L,L-2,6-diaminopimelate. Diaminopimelate epimerase E. colidapF1 (EC 5.1.1.7) generates meso-diaminopimelate fromL,L-diaminopimelate. Diaminopimelate decarboxylase E. coli lysA (EC4.1.1.20) generates L-lysine and CO2 from meso-diaminopimelate.

Alternatively, in lieu of dapD (EC 2.3.1.117), argD (EC 2.6.1.17), dapE(EC 3.5.1.18); LL-diaminopimelate aminotransferase A. thaliana At4g33680 (EC 2.6.1.83) is used to generate L,L-diaminopimelate andL-glutamate from tetrahydrodipicolinate and α-ketoglutarate. Homocitratesynthase S. cerevisiae lys21 (EC 2.3.3.14) generates homocitrate and CoAfrom acetyl-CoA and α-ketoglutarate. Homoaconitase S. cerevisiae lys4,lys3 (EC 4.2.1.36) generates homoisocitrate from homocitrate andhomo-cis-aconitate. Homoisocitrate dehydrogenase S. cerevisiae lys12,lys11, lys10 (EC 1.1.1.87) generates 2-oxoadipate and CO₂+NADH fromhomoisocitrate. 2-aminoadipate transaminase S. cerevisiae ARO8 (EC2.6.1.39) generates L-2-aminoadipate and α-ketoglutarate from2-oxoadipate and L-glutamate. 2-aminoadipate reductase S. cerevisiaelys2, lys5 (EC 1.2.1.31) generates L-2-aminoadipate 6-semialdehyde fromL-2-aminoadipate and NAD(P)H. Aminoadipate semialdehyde-glutamatereductase S. cerevisiae lys9, lys13 (EC 1.5.1.10) generatesN6-(L-1,3-Dicarboxypropyl)-L-lysine and NADP from L-glutamate andL-2-aminoadipate 6-semialdehyde and NADPH. Lysine-2-oxoglutaratereductase S. cerevisiae lys1 (EC 1.5.1.7) generates L-lysine andα-ketoglutarate and NADH from N6-(L-1,3-dicarboxypropyl)-L-lysine.

Example 34 Engineered Microorganisms Producing Serine

Phosphoglycerate dehydrogenase E. coli serA (EC 1.1.1.95) generates3-phosphonooxypyruvate and NADH from 3-P-D-glycerate. Phosphoserinetransaminase E. coli serC (EC 2.6.1.52) generates3-phosphonooxypyruvate+L-glutamate fromortho-P-L-serine+α-ketoglutarate. Phosphoserine phosphatase E. coli serB(EC 3.1.3.3) converts ortho-P-L-serine to L-serine.

Example 35 Engineered Microorganisms Producing Aspartate

Aspartate aminotransferase E. coli aspC (EC 2.6.1.1) convertsoxaloacetate and L-glutamate to L-aspartate and α-ketoglutarate.

Example 36 Engineered Microorganisms Producing Sorbitol

Sorbitol (from F6P) Glucose-6-phosphate isomerase E. coli pgi (EC5.3.1.9) converts D-β-fructose-6-P to D-α-glucose-6-P.

Phosphoglucomutase E. coli pgm (EC 5.4.2.2) converts D-α-glucose-6-P toD-α-glucose-1-P. Glucose-1-phosphatase E. coli agp (EC 3.1.3.10)converts D-α-glucose-1-P to D-α-glucose.

Alternatively, aldose-1-epimerase E. coli galM (EC 5.1.3.3) convertsD-β-glucose to D-α-glucose. Polyol dehydrogenase S. cerevisiae GRE3 (EC1.1.1.21) generates D-sorbitol from D-α-glucose and NADPH.

Example 37 Engineered Microorganisms Producing Ascorbate

Alpha-D-glucose-6-phosphate ketol-isomerase A. thaliana PGI1 (EC5.3.1.9) generates β-D-fructose-6-P from D-α-glucose-6-P.D-Mannose-6-phosphate ketol-isomerase A. thaliana din9 (EC 5.3.1.8)converts β-D-fructose-6-P to D-mannose-6-P. D-Mannose 6-phosphate1,6-phosphomutase A. thaliana atpmm (EC 5.4.2.8) converts D-mannose-6-Pto D-mannose-1-P. Mannose-1-phosphate guanylyltransferase A. thalianacyt (EC 2.7.7.22) converts D-mannose-1-P to GDP-mannose. GDP-mannose3,5-epimerase A. thaliana gme (EC 5.1.3.18) converts GDP-mannose toGDP-L-galactose. Galactose-1-phosphate guanylyltransferase A. thalianaVTC2 (EC 2.7.n.n) converts GDP-L-galactose to L-galactose-1-P.L-galactose 1-phosphate phosphatase A. thaliana VTC4 (EC 3.1.3.n)converts L-galactose-1-P to L-galactose. L-galactose dehydrogenase A.thaliana At4G33670 (EC 1.1.1.122) converts L-galactose toL-1,4-galactonolactone and NADH. L-galactonolactone oxidase S.cerevisiae ATGLDH (EC 1.3.3.12) converts L-1,4-galactonolactone and O₂to ascorbate and H2O2. A catalase E. coli katE (EC 1.11.1.6) (2H2O2=>O2)converts hydrogen peroxide to oxygen.

Example 38 Engineered Microorganisms Producing Cephalosporin

Homocitrate synthase S. cerevisiae lys21 (EC 2.3.3.14) convertsacetyl-CoA and α-ketoglutarate to homocitrate and CoA.

Homoaconitase S. cerevisiae lys4, lys3 (EC 4.2.1.36) generateshomocitrate or homo-cis-aconitate or homoisocitrate. Homoisocitratedehydrogenase S. cerevisiae lys12, lys1, lys10 (EC 1.1.1.87) generates2-oxoadipate and CO₂ and NADH from homoisocitrate. 2-aminoadipatetransaminase S. cerevisiae aro8 (EC 2.6.1.39) converts 2-oxoadipate andL-glutamate to L-2-aminoadipate and α-ketoglutarate. Phosphoglyceratedehydrogenase E. coli serA (EC 1.1.1.95) converts 3-P-D-glycerate to3-phosphonooxypyruvate and NADH. Phosphoserine transaminase E. coli serC(EC 2.6.1.52) converts ortho-P-L-serine and α-ketoglutarate to3-phosphonooxypyruvate and L-glutamate. Phosphoserine phosphatase E.coli serB (EC 3.1.3.3) converts ortho-P-L-serine to L-serine. SerineO-acetyltransferase A. thaliana AtSerat2; 1 (EC 2.3.1.30) convertsacetyl-CoA and L-serine to CoA and O-acetyl-L-serine. Cysteine synthaseA. thaliana At1G55880 (EC 2.5.1.47) converts O-acetyl-L-serine toL-cysteine and acetate. Acetolactate synthase E. coli ilvN, ilvB (EC2.2.1.6) converts pyruvate to CO2 and 2-acetolactate. Acetohydroxyacidisomeroreductase E. coli ilvC (EC 1.1.1.86) converts 2-acetolactate andNADPH to 2,3-dihydroxyisovalerate. Dihydroxyacid dehydratase E. coliilvD (EC 4.2.1.9) converts 2,3-dihydroxyisovalerate to2-ketoisovalerate. Valine transaminase E. coli ilvE (EC 2.6.1.42)converts 2-ketoisovalerate and L-glutamate to α-ketoglutarate andL-valine. ACV synthetase A. variabilis Ava_(—)1613 (EC 6.3.2.26)converts 3 ATP, L-2-aminoadipate, L-cysteine and L-valine toN-[L-5-amino-5-carboxypentanoyl]-L-cysteinyl-D-valine. Isopenicillin-Nsynthase A. variabilis Ava_(—)5009 (EC 1.21.3.1) convertsN-[L-5-amino-5-carboxypentanoyl]-L-cysteinyl-D-valine and O2 toisopenicillin-N. Isopenicillin-N epimerase M. xanthus cefD (EC 5.1.1.17)converts isopenicillin-N to penicillin-N. Cephalosporin biosynthesisexpandase/hydroxylase C. acremonium ceJEF (EC 1.14.20.1, 1.14.11.26)converts penicillin-N, 2α-ketoglutarate and 2 O2 todeacetylcephalosporin C, 2 succinate and 2 CO2. Deacetylcephalosporin-Cacetyltransferase C. acremonium cefG (EC 2.3.1.175) then generates CoAand cephalosporin C from acetyl-CoA and deacetylcephalosporin C.

Example 39 Engineered Microorganisms Producing Isopentenol

1-deoxy-D-xylulose-5-phosphate synthase E. coli dxs (EC 2.2.1.7)converts pyruvate and D-glyceraldehyde-3-P to 1-deoxy-D-xylulose-5-P andCO2. 1-deoxy-D-xylulose-5-phosphate reductoisomerase E. coli dxr (EC1.1.1.267) converts 1-deoxy-D-xylulose-5-P and NADPH to2-C-methyl-D-erythritol-4-P. 2-C-methyl-D-erythritol 4-phosphatecytidylyltransferase E. coli ispD (EC 2.7.7.60) converts CTP and2-C-methyl-D-erythritol 4-P to4-(cytidine-5′-PP)-2-C-methyl-D-erythritol. 4-(cytidine5′-diphospho)-2-C-methyl-D-erythritol kinase E. coli ispE (EC 2.7.1.148)converts ATP and 4-(cytidine-5′-PP)-2-C-methyl-D-erythritol to2-P-4-(cytidine 5′-PP)-2-C-methyl-D-erythritol. 2-C-methyl-D-erythritol2,4-cyclodiphosphate synthase E. coli ispF (EC 4.6.1.12) converts2-P-4-(cytidine 5′-PP)-2-C-methyl-D-erythritol to2-C-methyl-D-erythritol-2,4-cyclo-PP and CMP.4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase E. coli ispG (EC1.17.4.3) convert 2-C-methyl-D-erythritol-2,4-cyclo-PP to(E)-4-hydroxy-3-methylbut-2-en-1-yl-PP. 4-hydroxy-3-methylbut-2-enyldiphosphate reductase E. coli ispH (EC 1.17.1.2) convert(E)-4-hydroxy-3-methylbut-2-en-1-yl-PP and NADPH to isopentenyl-PP.4-hydroxy-3-methylbut-2-enyl diphosphate reductase E. coli ispH (EC1.17.1.2) converts (E)-4-hydroxy-3-methylbut-2-en-1-yl-PP and NADPH todimethylallyl-PP. Isopentenyl-diphosphate Δ-isomerase E. coli idi (EC5.3.3.2) converts dimethylallyl-PP to isopentenyl-PP. Isopentenyl-PPpyrophosphatase converts isopentenyl-PP to isopentenol. Isopentenoldikinase converts isopentenyl-PP to isopentenol and ATP.Hydroxymethylglutaryl-CoA synthase S. cerevisiae

erg13 (EC 2.3.3.10) converts acetyl-CoA and acetoacetyl-CoA to(S)-3-hydroxy-3-methylglutaryl-CoA and CoA. Hydroxymethylglutaryl-CoAreductase S. cerevisiae hmg2 (EC 1.1.1.34) converts (R)-mevalonate andCoA to (S)-3-hydroxy-3-methylglutaryl-CoA and 2 NADPH. Mevalonate kinaseS. cerevisiae erg12 (EC 2.7.1.36) converts ATP and (R)-mevalonate to(R)-5-P-mevalonate. Phosphomevalonate kinase S. cerevisiae erg8 (EC2.7.4.2) converts ATP and (R)-5-P-mevalonate to (R)-5-PP-mevalonate.Diphosphomevalonate decarboxylase S. cerevisiae mvd1 (EC 4.1.1.33)converts ATP and (R)-5-PP-mevalonate to isopentenyl-PP and CO2.

Example 40 Engineered Microorganisms Producing Lanosterol

1-deoxy-D-xylulose-5-phosphate synthase E. coli dxs (EC 2.2.1.7)converts pyruvate and D-glyceraldehyde-3-P to 1-deoxy-D-xylulose-5-P andCO2. 1-deoxy-D-xylulose-5-phosphate reductoisomerase E. coli dxr (EC1.1.1.267) converts 1-deoxy-D-xylulose-5-P and NADPH to2-C-methyl-D-erythritol-4-P. 2-C-methyl-D-erythritol 4-phosphatecytidylyltransferase E. coli ispD (EC 2.7.7.60) converts CTP and2-C-methyl-D-erythritol 4-P to4-(cytidine-5′-PP)-2-C-methyl-D-erythritol. 4-(cytidine5′-diphospho)-2-C-methyl-D-erythritol kinase E. coli ispE (EC 2.7.1.148)converts ATP and 4-(cytidine-5′-PP)-2-C-methyl-D-erythritol to2-P-4-(cytidine 5′-PP)-2-C-methyl-D-erythritol. 2-C-methyl-D-erythritol2,4-cyclodiphosphate synthase E. coli ispF (EC 4.6.1.12) converts2-P-4-(cytidine 5′-PP)-2-C-methyl-D-erythritol to2-C-methyl-D-erythritol-2,4-cyclo-PP and CMP.4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase E. coli ispG (EC1.17.4.3) converts 2-C-methyl-D-erythritol-2,4-cyclo-PP to(E)-4-hydroxy-3-methylbut-2-en-1-yl-PP. 4-hydroxy-3-methylbut-2-enyldiphosphate reductase E. coli ispH (EC 1.17.1.2) converts(E)-4-hydroxy-3-methylbut-2-en-1-yl-PP and NADPH to isopentenyl-PP.4-hydroxy-3-methylbut-2-enyl diphosphate reductase E. coli isp (EC1.17.1.2) converts(E)-4-hydroxy-3-methylbut-2-en-1-yl-PP+NADPH=dimethylallyl-PP.Isopentenyl-diphosphate Δ-isomerase E. coli idi (EC 5.3.3.2) convertsdimethylallyl-PP to isopentenyl-PP. Geranylgeranyl pyrophosphatesynthase Synechocystis sp. PCC6803 crtE (EC 2.5.1.29) convertsdimethylallyl-PP and 2 isopentenyl-PP to farnesyl-PP. Squalene synthaseSynechocystis sp. PCC6803 s110513 (EC 2.5.1.21) converts 2 farnesyl-PPand NADPH to squalene. Squalene monooxygenase S. cerevisiae erg1 (EC1.14.99.7) converts squalene, NADPH and O2 to (S)-squalene-2,3-epoxide.Lanosterol synthase S. cerevisiae ERG7 (EC 5.4.99.7) converts(S)-squalene-2,3-epoxide to lanosterol.

Example 41 Engineered Microorganisms Producing Omega-3 DHA

To engineer microorganisms producing omega-3 DHA, the necessary genesare pfaABCDE, some of which are multifunctional. Acetyl-CoA:ACPtransacylase S. pneumatophori (EC 2.3.1.38) converts acetyl-CoA and ACPto acetyl-ACP+CoA. Acetyl-CoA carboxylase E. coli (EC 6.4.1.2) convertsacetyl-CoA, ATP and CO2 to malonyl-CoA. Malonyl-CoA:ACP transacylase E.coli (EC 2.3.1.39) converts malonyl-CoA and ACP to malonyl-ACP and CoA.3-ketoacyl-ACP synthase E. coli (EC 2.3.1.41) converts acyl-ACP andmalonyl-ACP to CO2 and 3-ketoacyl-ACP. 3-ketoacyl-ACP reductase E. coli(EC 1.1.1.100) converts 3-ketoacyl-ACP and NADPH to 3-hydroxyacyl-ACP.3-hydroxyacyl-ACP dehydratase E. coli (EC 4.2.1.60) converts3-hydroxyacyl-ACP to enoyl-ACP. Enoyl-ACP reductase E. coli (EC 1.3.1.{9,10}) converts enoyl-ACP and {NADH, NADPH} to acyl-ACP. Desaturase S.pneumatophori (EC 1.14.19.n) converts m:n fatty acid, NADPH and O2 tom:(n+1) fatty acid. Acyl-ACP hydrolase S. cerevisiae FAS1 (EC 3.1.2.14)acyl-ACP to fatty acid and ACP.

Example 42 Engineered Microorganisms Producing Lycopene

1-deoxy-D-xylulose-5-phosphate synthase E. coli dxs (EC 2.2.1.7)converts pyruvate and D-glyceraldehyde-3-P to 1-deoxy-D-xylulose-5-P andCO2. 1-deoxy-D-xylulose-5-phosphate reductoisomerase E. coli dxr (EC1.1.1.267) converts 1-deoxy-D-xylulose-5-P and NADPH to2-C-methyl-D-erythritol-4-P. 2-C-methyl-D-erythritol 4-phosphatecytidylyltransferase E. coli ispD (EC 2.7.7.60) converts CTP and2-C-methyl-D-erythritol 4-P to4-(cytidine-5′-PP)-2-C-methyl-D-erythritol. 4-(cytidine5′-diphospho)-2-C-methyl-D-erythritol kinase E. coli ispE (EC 2.7.1.148)converts ATP+4-(cytidine-5′-PP)-2-C-methyl-D-erythritol to2-P-4-(cytidine 5′-PP)-2-C-methyl-D-erythritol. 2-C-methyl-D-erythritol2,4-cyclodiphosphate synthase E. coli ispF (EC 4.6.1.12) converts2-P-4-(cytidine 5′-PP)-2-C-methyl-D-erythritol to2-C-methyl-D-erythritol-2,4-cyclo-PP and CMP.4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase E. coli ispG (EC1.17.4.3) converts 2-C-methyl-D-erythritol-2,4-cyclo-PP to(E)-4-hydroxy-3-methylbut-2-en-1-yl-PP. 4-hydroxy-3-methylbut-2-enyldiphosphate reductase E. coli ispH (EC 1.17.1.2) converts(E)-4-hydroxy-3-methylbut-2-en-1-yl-PP and NADPH to isopentenyl-PP.4-hydroxy-3-methylbut-2-enyl diphosphate reductase E. coli ispH (EC1.17.1.2) converts (E)-4-hydroxy-3-methylbut-2-en-1-yl-PP and NADPH todimethylallyl-PP. Isopentenyl-diphosphate Δ-isomerase E. coli idi (EC5.3.3.2) converts dimethylallyl-PP to isopentenyl-PP. Geranylgeranylpyrophosphate synthase Synechocystis sp. PCC6803 crtE (EC 2.5.1.29)converts dimethylallyl-PP and 2 isopentenyl-PP to farnesyl-PP.Geranylgeranyl pyrophosphate synthase Synechocystis sp. PCC6803 crtE (EC2.5.1.29) converts isopentenyl-PP and farnesyl-PP to (alltrans)-geranylgeranyl-PP. Phytoene synthase Synechocystis sp. PCC6803crtB (EC 2.5.1.32) converts 2 geranylgeranyl-PP to phytoene. Phytoeneoxidoreductase Synechocystis sp. PCC6803 crtI (EC 1.14.99.n phytoene, 2NADPH and 2 O2 to ζ-carotene. ζ-carotene oxidoreductase Synechocystissp. PCC6803 crtQ-2 (EC 1.14.99.30) converts ζ-carotene, 2 NADPH and 2 O2to lycopene.

Example 43 Engineered Microorganisms Producing Itaconate

Aconitate decarboxylase from A. terreus (EC 4.1.1.6) convertscis-aconitate to itaconate and CO2. Itaconate can subsequently beconverted to various other carbon-based products of interest, e.g.,according to the scheme presented in FIG. 7.

Example 44 Engineered Microorganisms Producing 1,3-Butadiene orGlutamate

Succinyl-CoA dehydrogenase C. kluyveri sucD (EC 1.2.1.n) convertssuccinyl-CoA and NADPH to succinic semialdehyde and CoA.4-hydroxybutyrate dehydrogenase A. thaliana ghbdh (EC 1.1.1.2) convertssuccinic semialdehyde and NADPH to 4-hydroxybutyrate. Glutamatedehydrogenase E. coli gdhA (EC 1.4.1.4) converts α-ketoglutarate, NH3and NADPH to glutamate. Glutamate decarboxylase E. coli gadA (EC4.1.1.15) converts glutamate to 4-aminobutyrate and CO2. 4-aminobutyratetransaminase E. coli gabT (EC 2.6.1.19) converts 4-aminobutyrate andα-ketoglutarate to glutamate and succinic semialdehyde. Aldehydedehydrogenase E. coli aldH (EC 1.1.1.n) converts 4-hydroxybutyrate andNADH to 4-hydroxybutanal. 1,3-propanediol oxidoreductase K. pneumoniadhaT (EC 1.1.1.202)

-   4-hydroxybutanal and NADH to 1,4-butanediol. Alcohol dehydratase (EC    4.2.1.n) converts 1,4-butanediol to 1,3-butadiene.

Example 45 Engineered Microorganisms Producing Propylene

Acetyl-CoA carboxylase E. coli accBCAD (EC 6.4.1.2) converts acetyl-CoA,ATP and CO2 to malonyl-CoA. A bifunctional malonyl-CoA reductase C.aurantiacus (EC 1.2.1.18, 1.1.1.59) converts malonyl-CoA and 2 NADPH to3-hydroxypropionate and CoA. 3-hydroxypropionyl-CoA transferase C.kluyveri or fZ (EC 2.8.3.n) converts 3-hydroxypropionate and acetyl-CoAto 3-hydroxypropionyl-CoA and acetate. 3-hydroxypropionyl-CoAdehydratase C. aurantiacus (EC 4.2.1.17) converts 3-hydroxypropionyl-CoAto acryloyl-CoA. Acryloyl-CoA reductase C. aurantiacus (EC 1.3.1.n)converts acryloyl-CoA and NADPH to propionyl-CoA. PropionylCoA-transferase R. eutropha pct (EC 2.8.3.1) converts propionyl-CoA andacetate to acetyl-CoA and propionate. Aldehyde dehydrogenase E. coliadhE (EC 1.2.1. {3,4}) converts propionate and NADPH to propanal.Alcohol dehydrogenase E. coli adhE (EC 1.1.1. {1,2}) converts propanaland NADPH to 1-propanol. Alcohol dehydratase (EC 4.2.1.n) converts1-propanol to propylene.

Example 46 Engineered Microorganisms Producing Succinate, Citrate,Glutamate, Malate

From glyceraldehydes 3-phosphate (GAP), NAD+ and Pi, triosephosphatedehydrogenase converts GAP to 1,3-bisphosphoglycerate, NADH and H+.Phosphoglycerate kinase converts 1,3-bisphosphoglycerate and ADP to3-P-glycerate and ATP. Mutase converts 3-P-glycerate to 2-P-glycerate.Enolase converts 2-P-glycerate to phosphoenolpyruvate (PEP) and H2O.Phosphoenolpyruvate carboxylase then converts PEP to oxaloacetate (OAA).OAA is converted to succinate in one of two ways. OAA, H2O andacetyl-CoA are converted to citrate and CoASH by citrate synthase, whichconverts to H2O and cis-aconitate, an enzyme-bound intermediate, whichis subsequently converted to succinate by isocitrate lyase.Alternatively, cis-aconitate and H2O is converted to isocitrate byaconitase. Isocitrate and NADP+ are converted to oxalosuccinate, NADPHand H+ by isocitrate dehydrogenase. Oxalosuccinate is then converted toα-ketoglutarate and CO2 by isocitrate dehydrogenase. α-ketoglutarate,NAD+ and CoASH are converted to succinyl-CoA, CO2, NADH and H+ byα-ketoglutarate dehydrogenase. Succinate thiokinase convertsuccinyl-CoA, ADP and Pi to succinate, ATP and CoASH.

In those microorganisms where the above chemicals are already part ofcentral metabolism, they are engineered to export the chemicals from thecells. Under some conditions such as anaerobic fermentation, succinatecan build up in cells due to repression of the citric acid cycle. Oncethis occurs, one or more members of a family of enzymes known asC4-dicarboxylate carriers serve to export succinate from cells into themedia. Janausch et al., Biochimica et Biophysica Acta 1553:39-56 (2002);Kim et al., J. Bacteriol, March 2007, p. 1597-1603. In certain aspectsof the invention, succinate can be converted to various other chemicalsas illustrated in, e.g., FIG. 6.

Example 47 Analytical Methods to Detect 3-hpa

Colorimetric: To obtain standard curves, 0-6 mol of freshly distilledacrolein (Fluka, Buchs, Switzerland) is added to 6 ml of distilledwater. Then, 4.5 ml of DL-tryptophan (Fluka) solution (0.01 M solutionin 0.05 M HCl, stabilized with a few drops of toluene) and 18 ml of 37%HCl are added immediately. For 3-HPA quantification, a 1-ml sample ismixed with 0.75 ml of DL-tryptophan solution and 3 ml of HCl 37%.Mixtures containing samples and standards are incubated for 20 min in awater bath at 37° C. and the optical density is measured at 560 nm(OD560). 3-HPA samples are diluted with distilled water before mixingwith reagents to ensure a final OD560<1. This method is shown to allow aprecise quantification of 3-HPA using acrolein as a standard (Lüthi-Penget al. 2002a, b). The same tryptophan solution is used for the standardcurves and all 3-HPA quantifications and reported data are means forduplicate analyses. Appl Microbiol Biotechnol (2005) 68: 467-474

GC-MS: To determine the polyester content of the bacteria, 3 to 5 mg oflyophilized cell material is subjected to methanolysis in the presenceof 3 or 15% (v/v) sulfuric acid according to Brandl et al. (1988). Underthese conditions the intracellular poly(3-hydroxyalkanoates) aredegraded to their constituent 3-hydroxaylkanoic acid methyl esters. Themethyl esters are assayed by gas chromatography with a Perkin-Elmer 8420gas chromatograph equipped with a Permaphase PEG 25 Mx capillary column(25 m by 0.32 ram, Bodenseewerk Perkin Elmer, Uberlingen, FRG) and aflame ionization detector. A 2-1a1 portion of the organic phase isanalyzed after split injection (split ratio 1:40), and helium (35cm/min) is used as a carrier gas. The temperatures of the injector anddetector are 230° C. and 275° C., respectively. For efficient separationof the different 3-hydroxyalkanoic acid methyl esters the followingtemperature program is used: 120° C. for 5 min, temperature ramp of 8°C. per rain, 180° C. for 12 min. Calibration is performed withsynthesized methyl esters of standard 3-hydroxyalkanoic acids with 4 to12 carbon atoms (Brandl et al. 1988). Under these conditions theretention times of the different 3-hydroxyalkanoic acid methyl estersare as described recently (Timm et al. 1990). The total amount of PHAper cell dry weight is determined by summing the absolute amounts of allhydroxyalkanoate monomers detected. Arch Microbiol (1991) 155:415-421.

To determine the polymer content of lyophilized whole cells,approximately 4 mg of these cells is reacted in a small screw-cap testtube with a solution containing 1 ml of chloroform, 0.85 ml of methanol,and 0.15 ml of sulfuric acid for 140 min at 100° C. in athermostat-equipped oil bath (3; Lageveen, dissertation). This methoddegrades the intracellular PHA by methanolysis to its constituentP-hydroxycarboxylic acid methyl esters. After the reaction, 0.5 ml ofdistilled water is added and the tube is shaken vigorously for 1 min.After phase separation, the organic phase (bottom layer) is removed andtransferred to a small screw-cap glass vial. Samples are stored in thefreezer at −70° C. until further analysis. The methyl esters are assayedby gas chromatography (GC) with a Perkin-Elmer 8500 gas chromatographequipped with a Durabond-Carbowax-M15 megabore capillary column (CRnote: similar to DB-Wax column) (15 m by 0.54 mm; J & W Scientific) anda flame ionization detector. A 2-μl portion of the organic phase isanalyzed after splitless injection. Helium (17 ml/min) is used as thecarrier gas. The temperatures of the injector and detector are 230 and275° C., respectively. A temperature program is used which efficientlyseparated the different, -hydroxyalkanoic acid methyl esters (80° C. for4 min; temperature ramp of 8° C. per min; 160° C. for 6 min). Underthese conditions, the retention times of the different,B-hydroxyalkanoic acid methyl ester standards are as follows (min): C-4,4.22; C-5, 5.82; C-6, 7.40; C-7, 9.19; C-8, 10.71; C-10, 13.46; C-11,14.81; C-12, 16.61 (C-x represents the β-hydroxyalkanoic acid methylester with a chain length of x carbon atoms).

Example 48 Analytical Methods to Detect 1,3-PDL

The presence of 1,3-PDL determined by gas chromatography using a HP5890A equipped with a FID, a wide-bore column DB-5 (CR note: identicalresin to HP-5) (15 m×530 μm I.D.) with a film thickness of 1.5 μm. Theinstrumental conditions for calibration and assays are as follows:helium, hydrogen and air flow-rates were 8.5, 30 and 400 ml min-1,respectively; the injector port temperature is 220° C.; the detectortemperature was 260° C. The column is temperature-programmed from 40 to220° C. as follows: the initial temperature (40° C.) is held for 5 min,the temperature is then increased from 40 to 150° C. at 5° C. min-1, andheld for 1 min at 150° C., and then increased from 150 to 220° C. at 10°C. min-1. Samples are diluted 10 times with distilled water prior toinjection. See, e.g., Appl. Microbiol. Biotechnol., 59:289-296 (2002).

Example 49 Analytical Methods to Detect Succinate

Organic acids and glucose concentrations are determined by using aHewlett-Packard HPLC(HP 1090 series II) equipped with a UV monitor (210nm) and refractive index detector. Products are separated by using aBio-Rad HPX-87H column (10_(—)1 injection) with 4 mMH2SO4 as the mobilephase (0.4 ml_min_(—)1, 45° C.). Reference: T. B. Causey et al.,PNAS100: 825-832 (2003).

Example 50 Analytical Methods to Detect Lipids

FAME analysis coupled with direct injection GC/E1-MS quantification. Twoexample protocols:

4.5 ml of E. coli culture are acidified with 200 uL acetic acid,supplemented with 0.1 mg of pentadecanoic acid as an internal standard,and partitioned by adding 1:1 CHCl3:MeOH. The organic layer isevaporated to near dryness, resuspended in 1 ml 5% H2SO4 in MeOH, andincubated at 90 C for 2 h. The FAMEs are extracted with 300 uL Hexanesafter addition of 0.9% wt/vol NaCl in H2O. E1 GC-MS is performed on 1 uLof the Hexanes solution.

A different protocol that has been proposed to be “most-efficient” forlipid-producing bacteria involving treating freeze-dried cells at 90° C.for 60 min in the 3 ml mixture 10/1/1 v/v methanol/conc HCl/chloroform.1 ml water is then added, and the methyl esters are extracted byvortexing 3× with 2 ml 4/1 hexane/chloroform. (J. Microbiol. Meth. 2000,43, 107).

From 2 different cyanobacteria: Freshly harvested algal pellets (8 g)are boiled in 5 mL of isopropanol for 2 min to inhibit the lipaseactivity and are then dried under nitrogen gas. The dried pellet ishomogenized in chloroform-methanol (1:2 vol/vol) to achieve a finalvolume of 15 mL with 0.01% BHT added as an antioxidant in the lipidextraction solvent system. Lipid extract is centrifuged for 5 min at2000 g to remove cell debris. A total of 0.8 mL of distilled water isadded the to supernatant, followed by 5 mL of chloroform and 5 mL of0.88% potassium chloride in a separating funnel to achieve achloroform-methanol-water ratio of 1:1:0.9. The mixture is shakenvigorously for 5 min and allowed to separate for 30 min. The solventphase is collected and concentrated under nitrogen gas. The dried lipidextract is redissolved in 5.0 mL of chloroform and used for quantitativedetermination of different class of lipids. Fatty acid methyl esters areprepared for gas chromatograph (GC) analysis according to the protocolof Christie (20). The internal standard (1 mM heptadeconoic acid) isadded to the lipid sample and is subjected to methanolysis in thepresence of methanoic-HCl at 68-708° C. for 2 h. The methyl esters areextracted with three successive portions of hexane and treated with 5 mLof saturated solution of sodium bicarbonate and washed with 5 mL ofdistilled water. The upper (hexane) solution is evaporated to dryness ina water bath at 35-408° C. with the help of nitrogen gas. The methylesters are placed in a small volume of fresh hexane and 2 IL of samplewas injected into the injector port of the gas chromatograph (GC-Nucon).Photochemistry and Photobiology, 2006, 82: 702-710.

Example 51 Analytical Methods to Detect Amino Acids

From Waters website, supposedly the industry-standard amino aciddev/HPLC fluorescent detector combination: as described on the Waterswebsite under /WatersDivision/ContentD.asp?watersit=JDRS-5LTH9&WT.svl=1.

Derivitization: The AccQTag Method is based on a derivatizing reagentdeveloped specifically for amino acid analysis. Waters AccQFluor Reagent(6-aminoquinolyl-N-hydrozysuccinimidyl carbamate, or ACQ) is anN-hydroxysuccinimide-activated hetrocyclic carbamate, a new class ofamine-derivatizing compounds. Waters AccQFluor Reagent is a highlyreactive compound, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate(AQC), which forms stable derivatives with primary and secondary aminoacids in a matter of seconds. The derivatives are easily separated byreversed phase HPLC using Waters AccQTag Amino Acid Analysis System inless than 35 minutes. Excess reagent is consumed during the reaction toform aminoquinoline (AMQ). AMQ has significantly different spectralproperties than any of the derivatized amino acids, which allowsprogramming a detector wavelength that maximizes the spectral emissionresponse of the derivatives while minimizing the response of the AMQ.Detection limits for AQC-derivatized amino acids range from 38 to 320fmol. Fluorescence detection with 250 nm excitation, 395 nm emission orless sensitive UV detection at 248 nm.

GC-MS method: The labeling patterns of extracellular alanine, valine,and lysine are determined by GC-MS after conversion intot-butyl-dimethylsilyl (TBDMS) derivates withdimethyl-t-butyl-silyl-trifluororo-acetamide. For this purpose, 100 μlof cultivation supernatant is lyophilized. The freeze-dried residue isresuspended in 40 μl of dimethylformamide (0.1% pyridine) and 40 μl ofN-methyl-t-butyldimethylsilyltrifluoroacetamide (Macherey and Nagel,Easton, Pa.) and incubated at 80° C. for 1 h. GC-MS analysis is carriedout on a Hewlett-Packard 5890 series II gas chromatograph connected to aHewlett-Packard 5971 quadrupole mass selective detector (AgilentTechnologies, Waldbronn, Germany) with electron impact ionization at 70eV, and an RTX-5MS column (95% dimethyl-5% diphenylpolysiloxane; 30 m;320-μm inside diameter; Restek, Bellefonte, Pa.) is used with a columnhead pressure of 70 kPa and helium as the carrier gas. The columntemperature is initially kept at 120° C. for 5 min, subsequentlyincreased by 10° C./min up to 270° C., and maintained at thattemperature for 4 min. Other temperature settings are 270° C. (inlet),280° C. (interface), and 280° C. (quadrupole). For analysis, 1 μl ofsample is injected. TBDMS-derivatized alanine, valine, and lysine elutedafter 7, 12, and 22 min, respectively. All compounds exhibited a highsignal intensity for a fragment ion obtained by a mass loss of m-57 fromthe parent radical due to release of a t-butyl group from thederivatization residue. The fragment ions thus contain the entire carbonskeleton of the corresponding analyte. In order to increase thesensitivity, the mass isotopomer fractions m, m+1, and m+2 arequantified by selective ion monitoring of the corresponding ion clusterat m/z 260 to 262 (TBDMS-alanine), m/z 288 to 290 (TBDMS-valine), andm/z 431 to 433 (TBDMS-lysine). All measurements are carried out intriplicate. Ref: Applied and Environmental Microbiology, December 2002,p. 5843-5859, Vol. 68, No. 12. See Journal of Bioscience andBioengineering 2006, 102: 413-424, APPLIED AND ENVIRONMENTALMICROBIOLOGY, June 2007, p. 3859-3864 for other GC/MS analysis onextended set of amino acids, all but cysteine, tryptophan, glutamine andasparagines.

Example 52 Analytical Methods to Detect Lipids

General Photosynthetic Pigment Analysis (Ex Carotenoids) Cyanobacterialcells are collected after centrifugation of culture at 8000 g for 15min. The supernatant is discarded and the pellet of algal cells is driedin lyophilizer (Snijders, Holland). A total of 0.1 g of lyophilizedalgal cells are extracted in 1 mL 80% vol/vol methanol in a homogenizerat 48 C under dim light, followed by centrifugation at 6000 g for 10 minat 48° C. The samples are filtered through a 0.2 um filter before HPLCanalysis. The pigments are separated by HPLC with a reverse-phase column(Waters Spherisorb ODS, 25 lm34.6 mm3250 mm)(CR note: Agilent suggeststrying Zorbax SB-C18 column as substitute, 884950-567) and a PDAdetector (Waters 2996) according to the method described by Sharma andHall. A total of 20 μL of filtered sample is injected into the HPLC. Thegradient for separation is 0-100% ethyl acetate in acetonitrile-water(9:1 vol/vol) over 25 min with flow rate of 1.2 mL/min. The quantity ofpigments is calculated from peak area value using b-carotene as anexternal standard. Identification of pigments is performed by comparisonof the retention time against standard values and analysis of thespectral profile of individual peaks with a PDA detector in the range of400-700 nm. Photochemistry and Photobiology, 2006, 82: 702-710.

Example 53 Analytical Methods to Detect Lipids Phycobilisomes(Quantifying Phycocyanin, Allophycocyanin, Phycoerythrin)

Cell samples are concentrated by centrifugation for 15 min at 6000 g,0.1 g pellet is resuspended in 5 mL of 20 mM sodium acetate buffer (pH5.5) and cells are broken using sonicator (Bandelin UW 2200, Germany) at50% power with 9 cycles for 1 min. Phycobilisomes are precipitated byincubation with 1% streptomycin sulphate (wt/vol) for 30 min at 48 C andre collected by centrifugation at 8000 g for 30 min at 48 C. The amountof phycocyanin, allophycocyanin and phycoerythrin are calculatedaccording to the methods of Liotenberg et al. (15). Photochemistry andPhotobiology, 2006, 82: 702-710.

Example 54 Analytical Methods to Detect Erythromycin a (Bioassay andHPLC)

The titers of erythromycin produced by the industrial Saccharopolysporaerythraea strain are determined using a conventional bioassay withcommercially available erythromycin (Sigma) as a standard. Portions (20ml) of test medium (5 g peptone 1-1, 3 g beef extract 1-1, 3 g K2HPO41-1, and 15 g agar 1-1) are poured into Petri dishes (90 mm). Once themedium is solidified, a second layer consisting of 5-ml test medium with0.1% of a Bacillus pumilus [CMCC(B)6-3 202] is plated. ErA and itsgenetically engineered derivatives are extracted from culture broth anddetermined by HPLC according to Tsuji and Goetz (1978). Ref: Yong Wanget al. Improved production of erythromycin A by expression of aheterologous gene encoding S-adenosylmethionine synthetase. ApplMicrobiol Biotechnol (2007) 75:837-842.

A new HPLC-UV method for the determination of the impurity profile oferythromycin is developed. In contrast to the liquid chromatographydescribed in the European Pharmacopoeia the analysis could be performedat a temperature of 25° C. Erythromycin samples are analysed on anendcapped RP phase with cyanopropyl groups on the surface using gradientelution with 32 mM potassium phosphate buffer pH 8.0 andacetonitrile/methanol (75:25). The liquid chromatography forerythromycin is performed on an Agilent System 1100 LC (Boblingen,Germany) consisting of a vacuum degasser, a binary pumping systemforming a high pressure gradient by a static mixer (delay volume of600-900 μl), an autosampler, a thermostated column compartment, anUV-visible diode array detector (detection wavelength 215 nm) and a LC3D ChemStation equipped with HP Kayak XM600 and 3DSoftware (Version8.04). As a stationary phase, a Nucleodur CN-RP column (5 μm, 250 mm×4.0mm i.d.) (Macherey-Nagel, Düren, Germany) is used See, e.g., Deubel etal., Journal of Pharmaceutical and Biomedical Analysis, 43:493-498(2007).

Example 55 Engineered Microorganisms Producing Ethylene

Alcohol dehydratase (EC 4.2.1.n) converts ethanol to ethylene. Table 15,below, presents a list of alcohol dehydatases and their naturalsubstrates.

TABLE 15 EC number(s) Natural substrate 4.2.1.2 malate 4.2.1.3, 4.2.1.4citrate 4.2.1.11 2-phospho-D-glycerate 4.2.1.17, 4.2.1.553-hydroxybutyryl-CoA 4.2.1.33 3-isopropylmalate 4.2.1.34, 4.2.1.35{(R),(S)}-2-methylmalate 4.2.1.54 lactoyl-CoA 4.2.1.583-hydroxybutyryl-ACP 4.2.1.60 3-hydroxydecanoyl-CoA 4.2.1.68 L-fuconate4.2.1.74 hydroxyacyl-CoA 4.2.1.79 methylcitrate

Genes encoding ethylene-forming enzymes (EfE) from various sources,e.g., Pseudomonas syringae pv. Phaseolicola D13182, P. syringae pv. PisiAF 101061, Ralstonia solanacearum AL646053, may be expressed inmicroorganisms.

Construction of pJB5-efe_rs: The DNA sequence from the ethylene-formingenzyme of Ralstonia solanacearum (efe_rs) was obtained from Genbank(AL646053, (SEQ ID NO: 6); protein: CAD18680.1) and codon-optimized forE. coli (SEQ ID NO: 7). For examples of codon optimization of genes forE. coli, see Chandler et al., Mol. Plant, 1:285-94 (2008); Xue et al.,Enzyme and Microbial Technol. 42:58-64 (2007); and Chun et al., J Biol.Chem., 282:17486-500 (2007). All conflicting restriction sites used inthe pJB5 vector for cloning were removed from the gene to aid cloningexperiments. This optimized gene was obtained by contract synthesis fromDNA 2.0 (Menlo Park, Calif.). The efe_rs gene was designed with an NdeIsite including part of the start codon and an EcoRI site after the stopcodon. This gene was inserted by restriction digest with NdeI and EcoRI(New England Biolabs; Ipswitch, Mass.) on both pJB5 and the insertfollowed by ligation with a Quick Ligation Kit (New England Biolabs;Ipswitch, Mass.). The ligated construct was transformed into The NEB5-alpha F′Iq Competent E. coli (High Efficiency) (New England Biolabs:Ipswitch, Mass.).

Example 56 Engineered Microorganisms Producing Ethylene with pJB5-efe_rs

Genetically Modified Synechococcus sp. PCC 7002 (7002/efe_rs): Theconstruct as described in Example 55 was integrated onto the genome ofSynechococcus sp. PCC 7002 (Synechococcus 7002 or 7002) using thefollowing protocol. Synechococcus 7002 was grown for 48 h from coloniesin an incubated shaker flask at 30° C. at 2% CO2 to an OD₇₃₀ of 11n A⁺medium described in Frigaard N U et al. (2004) “Gene inactivation in thecyanobacterium Synechococcus sp. PCC 7002 and the green sulfur bacteriumChlorobium tepidum using in vitro-made DNA constructs and naturaltransformation” Methods Mol Biol 274:325-340. 900 μL of culture wasadded to a test-tube with 50 μL of 10 μg of DNA digested with xbaI ((NewEngland Biolabs; Ipswitch, Mass.) and added to cells without furtherpurification. DNA was prepped from a Qiagen Qiaprep Spin Miniprep Kit(Valencia, Calif.) for each construct. Cells were incubated in the darkfor four hours at 37° C. 100 L of cells were plated on A⁺ medium plateswith 1.5% agarose and grown to 30° C. for two days in low light. 10 g/mLof spectinomycin was underlayed on the plates. Resistant colonies werevisible in 7-10 days. 500 μL of cells remaining from the incubation withdigested DNA were added to 20 ml A+ cultures and bubbled with 1% CO2 atapproximately 1 bubble every 2 seconds for 24 h in the light. After 2 h,2 ml of the culture was transferred into 20 ml of A+ media containing 10μg/mL spectinomycin. After five days, the culture turned green and 1 mlwas passaged into 25 μg/mL spectinomycin and bubbled with 1% CO2 atapproximately 1 bubble every 2 seconds for 24 h in the light. After aperiod of 18 hours, the cells had achieved an OD₇₃₀=7.4 (dry weight 1.6g/L). 1 ml of this culture was placed in a 10 ml headspace vial (AgilentTechnologies) and 1 ml of a 7002 wild type culture was placed intoanother 10 ml headspace vial as a control. The two cultures wereincubated in a shaking incubator in the light for 1 h. The cultures werethen killed by incubating them at 80° C. for 5 min and analyzed for thepresence of ethylene.

Measurement of Ethylene by Headspace Gas Chromatography with FlameIonization Detection: Headspace-gas chromatography with flame-ionizationdetection (headspace GC-FID) can be used to analyze gases that areemitted from a liquid or solid sample that is contained inside anairtight vial capped with a septum. A sample of gas is obtained bypuncturing the septum with a syringe needle and then injecting the gassample into a temperature-controlled GC column. The mixture andsubsequently its individual components are carried through the column bya pressurized inert carrier gas that is flowing at a constant flow rate.Because different components of the mixture traverse the column atdifferent rates, they elute from the end of the column at differenttimes. When they emerge, they immediately enter the FID where a hydrogenflame ionizes them. This ionization yields a quantifiable electriccurrent which correlates with the amount of substance being ionized.Components can be identified by the amount of time they stay in thecolumn. This time is called the retention time.

Ethylene produced by a bacterial culture was analyzed. Because ofethylene's extremely low molecular weight (28.05) and minimal polarity,analysis of it by headspace GC requires that an appropriate column beused to separate it from other components in a gaseous mixture beingsubjected to headspace analysis. For this analysis, a J&W HP-PLOT/Qcapillary column with a length of 30 meters, a diameter of 0.53 mm, anda coating having a thickness of 40 micrometers was installed in a gaschromatograph (Agilent 7890A). The carrier gas was helium flowing at arate of 4.2 ml/minute. The column temperature was maintained at 60° C.The GC inlet where samples are injected into the column was maintainedat 150° C. with a split ratio of 20. The portion of the FID where thecomponents first elute from the column was maintained at 250° C.

Although headspace analysis can be done manually as described above,here an automated system (Agilent G1888) was used. The vial wastemperature equilibrated in an isothermal oven at 50° C. for 2 minuteswithout shaking. Subsequently the sample loop, which was maintained at60° C., was filled for 0.15 minutes and equilibrated for 0.1 minutes.Then the gas sample was transferred to the GC injection port for 0.5minutes through a transfer line maintained at 70° C.

Ethylene eluted from the column with a retention time of 2.52 minutes.This is in extremely good agreement with the retention time reported bythe column manufacturer (i.e., J&W) of 2.41 minutes using the sameconditions. The approximate amount of ethylene in the vials containing7002/efe_rs was 0.75 nanomoles/vial. FIG. 9 shows the GC/FIDchromatogram of the control 7002 strain. FIG. 10 shows the GC/FIDchromatogram of the 7002/efe_rs recombinant strain Ethylene wasquantified by using multiple headspace extraction [Kolb et al., StaticHeadspace-Gas Chromatography, 2^(nd) Ed, John Wiley & Sons (2006), pp.45-49] and the FID molar response factor (RF) for ethylene. The molar RFfor ethylene was extrapolated from the molar response factors for1-hexene, 1-heptene and 1-octene [Ackman, J Gas Chromatography, 6 (1968)497]. The response factors for those three compounds were measured usingmultiple headspace extraction.

Example 57 Engineered Microorganisms Producing Glucose

pJB336 was constructed in the following manner. A synthetic DNA kancassette (DNA2.0) was subcloned via flanking PacI and AscI restrictionsites into vector pJB303. This kan cassette comprises a promoter, activein both E. coli and JCC1, driving expression of gene aph that confersresistance to kanamycin in both organisms. pJB303 contains a syntheticDNA region (DNA2.0) comprising an upstream homology region (UHR) and adownstream homology region (DHR) flanking a multiple cloning region thatincludes PacI and AscI sites. The UHR corresponds to coordinates 1615342to 1615841, the DHR to coordinates 1617346 to 1617845, of the JCC1genome (Genbank Accession NC_(—)010475), respectively. The UHR and DHRmediate homologous recombinational integration of heterologous DNAflanked by these regions into the JCC1 chromosome. In the case ofSfiI-linearized pJB336, recombination occurs in such a way that the JCC1glgA1 gene encoding glycogen synthase 1 (SYNPCC7002-A1532; GenbankAccession YP_(—)001734779) is deleted, and replaced by a kan cassette.The SfiI-flanked DNA sequence contained within pJB336 is shown as SEQ IDNO: 8.

pJB342 was constructed in the following manner. A synthetic DNA speccassette (DNA2.0) was subcloned via flanking PacI and AscI restrictionsites into vector pJB301, creating vector pJB330. This spec cassettecomprises a promoter, active in both E. coli and JCC1, drivingexpression of gene aadA that confers resistance to spectinomycin andstreptomycin in both organisms. pJB301 contains a synthetic DNA region(DNA2.0) comprising an upstream homology region (UHR) and a downstreamhomology region (DHR) flanking a multiple cloning region that includesPacI and AscI sites. The UHR corresponds to coordinates 2207877 to2208376, the DHR to coordinates 2209929 to 2210428, of the JCC1 genome(Genbank Accession NC_(—)010475), respectively; the UHR and DHR mediatehomologous recombinational integration of heterologous DNA flanked bythese regions into the JCC1 chromosome. In parallel with theconstruction of pJB330, a synthetic TPT gene (DNA2.0) was subcloned viaflanking NdeI and EcoRI restriction sites into vector pJB 168, creatingvector pJB 171. TPT encodes a phosphate/triose-phosphate antiporttranslocator (UniProt Q9ZSR7) from Arabidopsis thaliana [Flügge U-I.,Ann. Rev. Plant Physiol. Plant Mol. Biol. 50:27-45 (1999)], and wascodon-optimized for expression in E. coli, checking for secondarystructure effects and removing restriction sites that were of utility inconstruct assembly strategies; the first seventy-seven amino acidsencoding the chloroplastic signal peptide were removed. This gene wasselected as it encodes a non-glucose transporter gene and thus serves asa negative control for assessing sugar transport. pJB 168 contains asynthetic DNA region (DNA2.0) comprising the E. coli lacI gene, encodingthe LacI repressor and driven by a lacI^(q) promoter, upstream of aLacI-repressed, IPTG-inducible P_(trc) promoter; in pJB 171, thispromoter controls expression of TPT. Via NotI and SpeI restrictionsites, the lacI/P_(trc)-TPT fragment of pJB 171 was then subclonedbetween the UHR and spec cassette of pJB330 to create plasmid pJB342. Inthe case of SfiI-linearized pJB342, recombination occurs in such a waythat the JCC1 glgA2 gene encoding glycogen synthase 2 (SYNPCC7002-A2125;Genbank Accession YP_(—)001735362) is deleted, and replaced by alacI/P_(trc)-TPT/spec cassette. The SfiI-flanked DNA sequence containedwithin pJB342 is SEQ ID NO: 9.

pJB345 was constructed in the following manner. A synthetic yihX gene(DNA2.0) was subcloned via flanking NdeI and EcoRI restriction sitesinto vector pJB 168, creating vector pJB 179. yihX encodes anα-D-glucose-1-phosphatase (UniProt P0A8Y3) from Escherichia coli K12(Kuznetsova E et al. (2006). Genome-wide Analysis of SubstrateSpecificities of the Escherichia coli Haloacid Dehalogenase-likePhosphatase Family. J. Biol. Chem. 281:36149-36161). A synthetic GLUT1gene (DNA2.0) was subcloned via flanking MfeI and SpeI restriction sitesinto vector pJB 179 (digested with EcoRI and SpeI), creating vector pJB185. GLUT1 encodes glucose transporter GLUT-1 (UniProt P11166) from Homosapiens (Zhao F-Q and Keating A F (2007). Functional Properties andGenomics of Glucose Transporters. Current Genomics 8:113-128), and wascodon-optimized for expression in E. coli, checking for secondarystructure effects and removing restriction sites that were of utility inconstruct assembly strategies. In plasmid pJB 185, a yihX-GLUT1 operonwas thus placed under the control of the P_(trc) promoter, itselfregulated by the upstream lacI gene. Via NotI and SpeI restrictionsites, the lacI/P_(trc)-yihX-GLUT1 fragment of pJB 185 was thensubcloned between the UHR and spec cassette of pJB330 to create plasmidpJB345. In the case of SfiI-linearized pJB345, recombination occurs insuch a way that the JCC1 glgA2 gene encoding glycogen synthase 2 isdeleted, and replaced by a lacI/P_(trc)-yihX-GLUT1/spec cassette. TheSfiI-flanked DNA sequence contained within pJB345 is shown as SEQ ID NO:10.

pJB348 was constructed in the following manner. A synthetic glf gene(DNA2.0) was subcloned via flanking MfeI and SpeI restriction sites intovector pJB 179 (digested with EcoRI and SpeI), creating vector pJB188.glf encodes glucose facilitated diffusion transporter Glf (UniProtP21906) from Zymomonas mobilis [Weisser P et al., J. Bacteriol.177:3351-3354 (1995)], and was codon-optimized for expression in E.coli, checking for secondary structure effects and removing restrictionsites that were of utility in construct assembly strategies. In plasmidpJB 188, a yihX-glf operon was thus placed under the control of theP_(trc) promoter, itself regulated by the upstream lacI gene. Via NotIand SpeI restriction sites, the lacI/P_(trc)-yihX-glf fragment of pJB188 was then subcloned between the UHR and spec cassette of pJB330 tocreate plasmid pJB348. In the case of SfiI-linearized pJB348,recombination occurs in such a way that the JCC1 glgA2 gene encodingglycogen synthase 2 is deleted, and replaced by a lacI/P_(trc)-yihX-glflspec cassette. The SfiI-flanked DNA sequence contained within pJB348 isshown as SEQ ID NO: 11.

Strain Construction: The UHR/DHR-flanked segments of pJB336, pJB342,pJB345, and pJB348 were integrated into the chromosome of JCC1 (pJB336)or JCC475 (pJB342, pJB345, and pJB348) in the following manner. Considerthe JCC1 transformation first. A culture of JCC1 was grown in A+ mediumto an OD₇₃₀ of 1 in a shaking incubator (Infors) at 37° C. in anatmosphere of 2% CO2 at 150 rpm. 0.5 ml of this culture was incubatedwith ˜5 g SfiI-digested plasmid pJB336-used without purificationfollowing the digest—for four hours at 37° C. in a low light (˜2 μE m²sec⁻¹ photosynthetically active radiation (PAR)) environment in ashaking incubator (250 rpm). The cell-DNA mixture was concentrated to 50μl and plated in its entirety onto an A⁺ agar plate, which wassubsequently incubated at 37° C. in a photoincubator (Percival; ˜50 μEm⁻² sec⁻¹ PAR) for approximately 24 hours in the absence of CO2enrichment. At this point, kanamycin was underlaid to a finalpost-diffusion concentration in the agar of 25 μg ml⁻¹, so as to selectfor integrants. Kanamycin-resistant colonies were visible afterapproximately five days, at which point the plates was transferred to a37° C. photoincubator (Percival; ˜50 μE m⁻² sec⁻¹ PAR) with a 1% CO2atmosphere, and incubated for a further two days.

To fully segregate recombinants, a small population (10-20) ofkanamycin-resistant colonies from this initial plate was streaked ontoan A+50 μg ml⁻¹ kanamycin plate, and grown as above; a small population(10-20) of kanamycin-resistant colonies from this second plate was thenstreaked onto an A+75 μg ml⁻¹ kanamycin plate, and grown as above.Genomic DNA was prepared from a single candidate JCC475 colony from thisthird plate and checked for complete segregation by PCR. For pJB336 andall other constructs, this involved checking for the presence of (i) theupstream recombinant junction, (ii) the downstream recombinant junction,and (iii) the heterologous gene(s), and the absence of the deletedwild-type gene (glgA1 or glgA2). Transformation of pJB342, pJB345, andpJB348 into JCC475 was carried out as described above except thatkanamycin was included in all plates to maintain selection for theglgA1::kan disruption in JCC475, and spectinomycin was used as theselective antibiotic—first at 25 μg ml⁻¹, then at 50 μg ml⁻¹, andfinally at 75 μg m⁻¹.

In this way, the ΔglgA1::kan ΔglgA2::spec JCC1-derived strains shown inTable 16 were constructed:

TABLE 16 ΔglgA1::kan ΔglgA2::spec JCC1-derived recombinant strains usedfor sugar production Transforming Integration Heterologous Strain ParentStrain DNA locus Promoter gene(s) Marker(s) JCC475 JCC1 (Synechococcussp. pJB336 ΔglgA1 — — Kan PCC 7002) JCC342c JCC475 pJB342 ΔglgA2lacI/P_(trc) TPT kan, (control) spec JCC543, JCC475 pJB345 ΔglgA2lacI/P_(trc) yihX- kan, JCC545 GLUT1 spec JCC547 JCC475 pJB348 ΔglgA2lacI/P_(trc) yihX-glf kan, spec

In reference to the above table, JCC543 and JCC545 representindependently isolated colonies from the final segregation plate; twowere selected to assess the genotypic reproducibility of any sugarproduction phenotype that emerged. JCC543, JCC545, and JCC547 werecompletely and faithfully segregated as determined by PCR in that theyyielded the expected upstream junction, downstream junction, andheterologous gene(s) amplicons, as well as lack of an ampliconcorresponding to glgA2-internal sequence (unlike JCC1). While JCC342cfailed, as anticipated, to give an amplicon corresponding to glgA2, ityielded only the downstream junction amplicon, indicating that whileglgA2 had been successfully deleted in this strain and the spec cassettewas in the expected location relative to the DHR, the lacI/P_(trc)-TPTregion had been somehow corrupted, most likely due to the toxicity ofeven small levels of TPT expression. Despite this, JCC342c, by virtue ofbeing ΔglgA1::kan ΔglgA2::spec and having been made in the same way andat the same time as JCC543, JCC545, and JCC547, served as an idealnegative control for determining whether these three strains were ableto export, or augment export of, sugar(s) into the medium. Note that allstrains, because they lack all genes encoding glycogen synthases, weredesigned to be unable to produce glycogen.

Sugar Production: Glucose was assayed in the culture medium of JCC342c,JCC543, JCC545, and JCC547 both enzymatically and by gas chromatographymass spectrometry (GC-MS). These two methods, which give concordantresults, are treated separately below.

Enzymatic assay: A single colony of each of JCC342c, JCC543, JCC545, andJCC547 was inoculated into 10 ml A+ medium containing 75 μg/ml kanamycinand 75 μg/ml spectinomycin. These cultures were incubated at 37° C. forapproximately three days, slowly and continuously bubbled with airenriched with 1% CO₂ in ˜50 μE m⁻² sec⁻¹ PAR. Cells were washed twicewith 10 ml A+ medium containing 75 μg/ml kanamycin, 75 μg/mlspectinomycin, and 0.5 mM IPTG, and seeded into 30 ml cultures of thesame medium at an initial OD₇₃₀ of 0.07. These cultures were incubatedin a shaking photoincubator (Infors) at 150 rpm at 37° C. for 15 days ina 2% CO₂ atmosphere and continuous light (˜100 μE m⁻² sec⁻¹ PAR). Waterlost by evaporation was replaced every two days with the appropriatevolumes of sterile Milli-Q water. 0.2 ml of culture was sampled on days7, 11, 12, and 15; cells were pelleted by centrifugation, and theculture supernatant was frozen at −20° C. until ready to be assayed forglucose. Culture supernatants were all assayed at the same time.

The Maltose and Glucose Assay Kit (Biovision; catalog number K618-100)was used to determine the concentration of glucose in culturesupernatant. In a flat-bottomed 96-well plate well, 10 μl of culturesupernatant was mixed with 86 μl Glucose Assay Buffer (GAB; K618-100-1),2 μl DMSO-dissolved Glucose Probe (K618-100-2), and 2 μl GAB-dissolvedGlucose Enzyme Mix (K618-100-5). These 100 μl reaction mixtures wereincubated for 1 hour at 37° C. in the dark. OD₅₇₀ was then measuredusing a microplate reader (SpectraMax). To relate OD₅₇₀ to absoluteD-glucose concentrations, at the same time as the above reactionsmixtures were being assembled, 10 μl of solutions of knownconcentrations of D-glucose from 0 to 54 mg liter⁻¹ dissolved in A+medium were assayed in the same fashion. The OD₅₇₀ measurements for theculture supernatants were thereby converted to D-glucose concentrations.

As shown in Table 17, JCC543 and JCC545, the replicate ΔglgA1::kanΔglgA2::lacI-P_(trc)-yihX-GLUT1-spec strains, produced five times moreglucose than the control ΔglgA1::kan ΔglgA2::spec JCC342c strain, whenall three cultures were at a comparable cell density (OD₇₃₀ 13-15).JCC547, the ΔglgA1::kan ΔglgA2::lacI-P_(trc)-yihX-glf-spec strain, grewsignificantly more slowly than the other three strains, failing to growbeyond OD₇₃₀ 9.6 during the course of the experiment; at this OD₇₃₀, theculture supernatant of JCC547 had a glucose concentration comparable tothat of JCC342c.

TABLE 17 Glucose produced in the culture media of JCC342c, JCC547,JCC543, and JCC545 as determined by an enzymatic assay Cell Glucose inDensity Culture Medium Strain Genotype (OD₇₃₀) (mg/liter) JCC342cΔglgA1::kan ΔglgA2::spec 15.0 9 JCC547 ΔglgA1::kan ΔglgA2::lacI- 9.6 6P_(trc)-yihX-glf-spec JCC543 ΔglgA1::kan ΔglgA2::lacI- 13.1 52P_(trc)-yihX-GLUT1-spec JCC545 ΔglgA1::kan ΔglgA2::lacI- 13.8 53P_(trc)-yihX-GLUT1-spec

GC-MS assay: In a separate growth experiment to the one described in theEnzymatic Assay section, a single colony of each of JCC342c, JCC543,JCC545, and JCC547 was inoculated into 10 ml A+ medium containing 75μg/ml kanamycin and 75 μg/ml spectinomycin. These cultures wereincubated at 37° C. for approximately three days, slowly andcontinuously bubbled with air enriched with 1% CO₂ in ˜50 μE m⁻² sec⁻¹PAR. Cells were washed twice with 10 ml A+ medium containing 75 μg/mlkanamycin, 75 μg/ml spectinomycin, and 0.5 mM IPTG, and seeded into 30ml cultures of the same medium at an initial OD₇₃₀ of 0.05. Thesecultures were incubated in a shaking photoincubator (Infors) at 150 rpmat 37° C. for 8 days in a 2% CO₂ atmosphere and continuous light (˜100μE m⁻² sec⁻¹ PAR). Two milliliters of culture was sampled on the finalday. Cells were pelleted by centrifugation, and the culture supernatantwas filtered through a 0.2 μm filter to remove any remaining cells. 0.9ml of filtered culture supernatant was lyophilized overnight inpreparation of derivatization for GC-MS.

Lyophilized residue was partially dissolved in 200 μl of anhydrouspyridine by vigorous vortexing to which was added 1.0 ml of silylatingreagent (BSA+TMCS+TMSI, 3:2:3; Supelco, Bellefonte, Pa.). Mixtures weresubjected to substantial vortexing and then placed at 70° C. for twohours with occasional vortexing. After cooling to room temperature, thederivatized sample was transferred to a glass autosampler vial inpreparation for GC-MS analysis using an Agilent 7890A GC equipped with a5975C electron-impact MS. 1.0 μl of derivatized sample was injected intothe GC with a 7683 automatic liquid sampler equipped with a 10 μlsyringe. The GC inlet temperature was 280° C. and a split ratio of 5 wasused. The capillary column was an Agilent HP-5MS (30 m×0.25 mm×0.25 μm).The carrier gas was helium at a flow rate of 1.0 ml min⁻¹. The GC oventemperature program was 50° C., hold 1 min; 10° C. min⁻¹ to 280° C.,hold 10 min. The GC-MS interface temperature was 290° C., the massspectrometer source temperature was 230° C. and the quadrupoletemperature was 150° C. The mass range was 25-1000 amu. Sugar peakspresent in the total ion chromatograms were identified by theirretention times by using authentic standards (Sigma-Aldrich) and bysearching an NIST MS database (2008 version).

As shown in Table 18, JCC543 and JCC545 had produced over twice as muchglucose than the control JCC342c strain, despite the former being atlower cell density (OD₇₃₀ 9.7 and 10.8) than the latter (OD₇₃₀ 15.1).JCC547 produced basal amounts of glucose. FIG. 16 shows the total ionchromatogram (TIC) for JCC342c, JCC545, and JCC547 in the retention timewindow during which TMS-derivatized α-D-glucose and β-D-glucose elute,from which the combined peak areas in Table 18 were derived.

TABLE 18 Glucose produced in the culture media of JCC342c, JCC547,JCC543, and JCC545, as determined by total ion chromatogram peak areasfor glucose seen by GC-MS analysis Cell Combined TIC peak Density areafor α- and Strain Genotype (OD₇₃₀) β-D-glucose JCC342c ΔglgA1::kanΔglgA2::spec 15.1 2341996 JCC547 ΔglgA1::kan ΔglgA2::lacI- 7.2 1581200P_(trc)-yihX-glf-spec JCC543 ΔglgA1::kan ΔglgA2::lacI- 9.7 5827988P_(trc)-yihX-GLUT1-spec JCC545 ΔglgA1::kan ΔglgA2::lacI- 10.8 6114673P_(trc)-yihX-GLUT1-spec

These GC-MS data corroborate the independent enzymatic assay datareported in the previous section, namely that the P_(trc)-yihX-GLUT1cassette, but not the P_(trc)-yihX-glf cassette in JCC547, resulted insignificantly higher glucose production than observed in an isogeniccontrol strain.

Although GC-MS analysis indicated only basal levels of glucose werepresent in the growth medium of JCC547, it was apparent that there wereseveral other ion chromatogram peaks only present, or present withlarger areas, in this strain and not in JCC342c, JCC543, or JCC545. Oneof these peaks was positively identified as sucrose based on authenticstandard analysis, as shown in Table 19 and FIG. 17. Based on theconcentration of sucrose used in the authentic standard analysis, andassuming that the TIC peak area observed scales linearly with this knownsucrose concentration, it was estimated that the JCC547 culture mediumcontained approximately 600 mg liter⁻¹ sucrose, approximately 100 timesthat seen in JCC342c, JCC543, or JCC545. No maltose was observed in anyof the four strains' culture media.

TABLE 19 Sucrose produced in the culture media of JCC342c, JCC547,JCC543, and JCC545, as determined by extracted ion chromatogram peakareas for the m/z 361 diagnostic disaccharide ion seen by GC-MS analysisExtracted ion chromatogram (EIC) peak area for sucrose Strain Genotype(m/z = 361 ion) JCC342c ΔglgA1::kan ΔglgA2::spec 63632 JCC547ΔglgA1::kan ΔglgA2::lacI- 8892575 P_(trc)-yihX-glf-spec JCC543ΔglgA1::kan ΔglgA2::lacI- 50666 P_(trc)-yihX-GLUT1-spec JCC545ΔglgA1::kan ΔglgA2::lacI- 53940 P_(trc)-yihX-GLUT1-spec

The P_(trc)-yihX-glf cassette in JCC547 therefore resulted insignificantly higher sucrose production than observed in an isogeniccontrol strain. A possible reason for this is that the Glf transporteris able to mediate the export of sucrose that is naturally synthesized,and otherwise maintained, within the cell. There are no reports of Glfbeing able to mediate transport of disaccharides such as sucrose. Glfhas been reported as being able to mediate transport of glucose and, toa much lesser degree, fructose. However, the notion of Glf-mediateddisaccharide export was supported by the GC-MS analysis of the culturemedium of JCC547. As mentioned above, GC-MS indicated several ionchromatogram peaks that were only present, or present with larger areas,in this strain and not in JCC342c, JCC543, or JCC545. Consistent withthese peaks representing disaccharides, many of these peaks werecharacterized by a dominant m/z 361 ion, which is diagnostic ofTMS-derivatized disaccharides [Molnár-Perl et al., Chem. Mater. Sci.,45:321-327 (1997)], as shown in Table 20. Because none of the authenticdisaccharide standards that were used eluted at the times indicated inthe table above, none of these corresponding peaks could be identifiedwith certainty. However, given the presence of the m/z 361 ion in all,it is highly likely that these peaks represent disaccharide ordisaccharide-like molecules, most likely synthesized naturally withinthe cell.

TABLE 20 Disaccharide and/or disaccharide-like molecules produced byJCC547, as determined by extracted ion chromatogram peak areas for them/z 361 diagnostic disaccharide ion seen by GC-MS analysis* EIC peakarea for diagnostic m/z = 361 ion at the following elution times (min)Strain Genotype 20.41 21.30 21.84 21.98 22.07 22.15 22.56 22.77 JCC342cΔglgA1::kan 0 0 0 0 0 0 0 0 ΔglgA2::spec JCC547 ΔglgA1::kan 6063564499225 193663 46991 114604 91013 65186 14880 ΔglgA2::lacI-P_(trc)-yihX-glf- spec JCC543 ΔglgA1::kan 0 0 0 0 0 0 0 0 ΔglgA2::lacI-P_(trc)-yihX- GLUT1-spec JCC545 ΔglgA1::kan 0 0 0 0 0 0 0 0ΔglgA2::lacI- Ptrc-yihX- GLUT1-spec *These peaks have not beenassociated definitively with defined chemical species.

Example 58 Engineered Microorganisms Producing Maltose with AmylaseExpressing Plasmids

Construction of amylase expressing plasmids: The DNA sequence of theamylase genes of Glycine max (AMY_gm), Bacillus cereus (AMY_bc), and thetransporter genes from Arabidopsis thaliana (MEX1) and Escherichia coli(setA) were obtained from Genbank. The codon sequences were optimizedfor E. coli. The codon-optimized sequences and amino acid sequences forBAA34650 (SEQ ID NO: 12 and SEQ ID NO:13, respectively), CAA50551 (SEQID NO: 14 and SEQ ID NO: 15, respectively), AAF04350 (SEQ ID NO: 16 andSEQ ID NO: 17, respectively), and YP_(—)025293 (SEQ ID NO: 18 and SEQ IDNO: 19, respectively) are provided herein. The DNA sequence of aphII,amt2, and trc promoters were obtained from Genbank and codon-optimizedfor E. coli (SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22,respectively). The trc promoter was engineered with an upstream lacI^(q)gene that is constitutively expressed. For examples of codonoptimization of genes for E. coli, see Chandler et al.,“Characterization of Gibberellin receptor mutants of Barley (Hordeumvulgare L.)”, Mol. Plant. 2008 1:285-94; Xue et al., Improved productionof p-hydroxycinnamic acid from tyrosine using a novel thermostablephenylalanine/tyrosine ammonia lyase enzyme, Enzyme and MicrobialTechnol. 2007 42:58-64.; and Chun et al., Electron transport pathway fora Streptomyces cytochrome P450: cytochrome P450 105D5-catalyzed fattyacid hydroxylation in Streptomyces coelicolor A3(2). J Biol. Chem. 2007282:17486-500. These optimized genes were obtained by contract synthesisfrom DNA 2.0 (Menlo Park, Calif.). In addition, plasmids containing two750 bp regions of homology designed to remove the native glgB (pJB315)or the restriction site (pJB318) from Synechococcus sp. PCC 7002 wereobtained by contract synthesis from DNA 2.0 (Menlo Park, Calif.). UsingpJB315 and pJB318 as vectors, the constructs were engineered byperforming 4 sequential clonings: insertion of aadA using PacI and IAscI, insertion of the amylase/transporter cassette using NdeI andEcoRI, insertion of the promoter-cat cassette with NotI and NdeI, andremoval of the cat gene using SfiI. All restriction and ligation enzymeswere obtained from New England Biolabs (Ipswich, Mass.). Ligatedconstructs were transformed into either NEB 5-α competent E. coli (HighEfficiency) (New England Biolabs: Ipswich, Mass.) or Copy Cutter EPI400competent E. coli (Epicentre Biotechnologies: Madison, Wis.).

Genetically Modified Synechococcus Sp. PCC 7002(7002/Amylase_Transporter): The constructs as described above wereintegrated onto the genome of Synechococcus sp. PCC 7002 (Synechococcus7002 or 7002) using the following protocol. Synechococcus 7002 was grownin an incubated shaker flask at 37° C. at 1% CO₂ to an OD₇₃₀ of 1.2 inA⁺ medium described in Frigaard N U et al., Methods Mol. Biol.,274:325-340 (2004). 1000 μL of culture was added to a test-tube with 50μL of 2 μg of DNA digested with XbaI (New England Biolabs; Ipswich,Mass.) and added to cells without further purification. DNA was preppedfrom a Qiagen Qiaprep Spin Miniprep Kit (Valencia, Calif.) for eachconstruct. Cells were incubated in the dark for two hours at 37° C. Theentire volume of cells were plated on A⁺ medium plates with 1.5% agaroseand grown to 37° C. in a lighted incubator (40-60 μE/m2/s PAR, measuredwith a LI-250A light meter (LI-COR)) for approximately 24 hours. 25μg/mL of spectinomycin was underlayed on the plates. After furtherincubation, resistant colonies became visible in 5 days. One colony fromeach of the 18 cultures (JCC724-741) was restreaked onto A⁺ mediumplates with 1.5% agarose and 50 g/mL spectinomycin. Colonies from theseplates were then inoculated into 5 ml of A+ media containing 25 μg/mlspectinomycin. This culture was incubated in a bubble tube at 37° C.sparged at approximately 1-2 bubbles of 1% CO₂/air every 2 seconds inlight (40-50 μE/m2/s PAR, measured with a LI-250A light meter (LI-COR)).Strains containing amylase-transporter constructs under constitutiveexpression were harvested at OD₇₃₀ ranging from 1.04 to 9.44. Strainscontaining amylase-transporter constructs under trc expression wereincubated to OD₇₃₀ between 3.76 and 8.15. 1 mL of these uninducedcultures was harvested. The remaining cultures were diluted to OD₇₃₀ of1, induced with 0.05 mM IPTG, and incubated for 24 hours. To harvestcells, cultures were spun for 1 minute at 14800 rpm. The supernatant wassubsequently submitted for GC/MS analysis.

Measurement of maltose by gas chromatography: Samples were partiallydissolved in 300 μL anhydrous pyridine (Sigma Aldrich; St Louis Mo.)before adding 1.0 mL of silylation reagent (BSA+TMCS+TMSI 3:2:3(Supelco; Bellefonte Pa.)). After each addition, samples were subjectedto vigorous vortexing. Samples were then heated at 70° C. for two hours,cooled, transferred to autosampler vials, and measured with the GC/MS.Retention time in minutes of α-maltose and β-maltose were identified bytheir mass spectra and their retention times.

An Agilent 7890A GC/5975C E1-MS equipped with a 7683 series autosamplerwas used to detect maltose. The GC inlet was set to a split ratio of 5.0and the inlet temperature was set to 280° C. 1 μL of sample was injectedinto a HP-5MS column (Agilent, 30 m×0.25 mm×0.25 μm). The carrier gaswas helium. The GC oven temperature program was 50° C., hold one min;10°/min increase to 280° C., hold ten min. The GC-MS interface was setto 290° C., and the MS mass range was 25 to 1000 amu. Peaks present inthe total-ion chromatograms were identified by retention time analysisand by searching the NIST MS Search database version 2.0 (2008) with theassociated mass spectra. Retention times in minutes of α-maltose (24.73min) and β-maltose (25.06 min) were determined with authentic standardsfrom Fluka and are represented in FIG. 18.

Maltose was detected in JCC726, 729, 735, and 738 (FIG. 19). Maltose wasproduced only in strains in which expression of the amylase-transporteroperon was controlled by the trc promoter.

Example 59 Engineered Methanogenesis Pathway

A host cell of interest is engineered to produce methane. Preferably, ahost is selected or engineered to have methanogenic properties.

TABLE 21 Example Organism, Enzyme EC No. gene(s) formylmethanofuran1.2.99.5 Methanosarcina dehydrogenase acetivorans fmdEFACDBformylmethanofuran- 2.3.1.101 Methanosarcina tetrahydromethanopterinacetivorans ftr formyltransferase methenyltetrahydromethanopterin3.5.4.27 Methanosarcina cyclohydrolase acetivorans mchmethylenetetrahydromethanopterin 1.5.99.9 Methanosarcina dehydrogenaseacetivorans mer 5,10- 1.5.99.11 Methanococcusmethylenetetrahydromethanopterin maripaludis hmd reductasetetrahydromethanopterin S- 2.1.1.86 Methanosarcina methyltransferaseacetivorans mtrHGFABCDE methyl-coenzyme M reductase 2.8.4.1Methanosphaera stadtmanae mrtBGA heterodisulfide reductase 1.8.98.1Methanococcus aeolicus Maeo_0307 coenzyme F420 hydrogenase 1.12.98.1Methanococcus maripaludis frcBGDA

Example 60 Engineered Acetogenesis Pathway

A host cell of interest is engineered to produce acetate. Preferably,the host cell is selected or engineered to have acetogenic properties.

TABLE 22 Example Organism, Enzyme EC No. gene(s) Phosphotransacetylase2.3.1.8 Escherichia coli pta Acetate kinase 2.7.2.1 Escherichia coliackA formate dehydrogenase 1.2.1.2 Escherichia coli fdhFformyltetrahydrofolate 6.3.4.3 Clostridium synthetase acetobutylicumCAC3201 5,10-methylenetetrahydrofolate 3.5.4.9 Escherichia colicyclohydrolase folD (bifunctional) 5,10-methylenetetrahydrofolate1.5.1.5 Escherichia coli dehydrogenase folD (bifunctional)methylenetetrahydrofolate 1.5.1.20 Arabidopsis reductase [NAD(P)H]thaliana MTHFR1 Methyltransferase 2.1.1.— Clostridium thermoaceticumCarbon monoxide 1.2.99.2 Clostridium dehydrogenase beijerinckiiCbei_3020 Hydrogenase — Escherichia coli hycDCFGBE

Example 61 Engineered Reductive TCA Cycle

A host cell of interest is engineered to have a reductive TCA cycle.Preferably, the host cell is selected or engineered to havephotosynthetic properties.

TABLE 22 Example Organism, Enzyme EC No. gene(s) PEP carboxylase4.1.1.31 Escherichia coli ppc α-ketoglutarate synthase 1.2.7.3Campylobacter jejuni oorDABC aconitase 4.2.1.3 Escherichia coli acnBisocitrate dehydrogenase 1.1.1.42 Escherichia coli (NADP+) icdcitrate-ATP lyase 2.3.3.8 Arabidopsis thaliana ACLB-1 fumarate reductase1.3.99.1 Escherichia coli frdDCBA fumarate hydratase 4.2.1.2 Escherichiacoli fumCAB malate dehydrogenase 1.1.1.37 Escherichia coli mdhsuccinate-CoA ligase 6.2.1.5 Escherichia coli (ADP-forming) sucCDpyruvate: ferredoxin 1.2.7.1 Clostridium oxidoreductase kluyveri porBPEP synthetase 2.7.9.2 Escherichia coli pps

Example 62 Engineered Calvin Cycle

A host cell of interest is engineered to have a Calvin cycle.Preferably, the host cell is selected or engineered to havephotosynthetic properties.

TABLE 23 Example Organism, Enzyme EC No. gene(s)D-ribulose-1,5-bisphosphate 4.1.1.39 Arabidopsis carboxylase thalianarbcL, ATS1A, ATS1B, ATS2B phosphoketolase 4.1.2.9 Pseudomonas putidaPput_2459 fructose-bisphosphate 4.1.2.13 Escherichia coli aldolase fbaBAtransketolase 2.2.1.1 Escherichia coli tktBA fructose-bisphosphatase3.1.3.11 Escherichia coli fbp sedoheptulose- 3.1.3.37 Arabidopsisbisphosphatase thaliana SBPASE triose-phosphate isomerase 5.3.1.1Escherichia coli tpiA glyceraldehyde-3-phosphate 1.2.1.13 Arabidopsisdehydrogenase (NADP+) thaliana GAPA (phosphorylating);ribose-5-phosphate 5.3.1.6 Escherichia coli isomerase rpiAribulose-phosphate 5.1.3.1 Escherichia coli 3-epimerase rpephosphoglycerate kinase 2.7.2.3 Escherichia coli pgk phosphoribulokinase2.7.1.19 Arabidopsis thaliana PRK

Example 63 Engineered 3-HPA Cycle

A host cell of interest is engineered to have a 3-HPA cycle. Preferably,the host cell is selected or engineered to have photosyntheticproperties.

TABLE 24 Example Organism, Enzyme EC No. gene(s) Acetyl-CoA carboxylase6.4.1.2 Chloroflexus aurantiacus malonatesemialdehyde 1.2.1.18Chloroflexus aurantiacus dehydrogenase (NADP− acylating)3-hydroxypropionate 1.1.1.— Chloroflexus aurantiacus dehydrogenase(NADP+) 3-hydroxypropionate-CoA 6.2.1.— Chloroflexus aurantiacus ligaseacryloyl-CoA hydratase 4.2.1.— Chloroflexus aurantiacus acryloyl-CoAreductase 1.3.1.— Chloroflexus aurantiacus (NADPH) propionyl-CoAcarboxylase 6.4.1.3 Chloroflexus aurantiacus methylmalonyl-CoA epimerase5.1.99.1 Chloroflexus aurantiacus methylmalonyl-CoA mutase 5.4.99.2Chloroflexus aurantiacus succinyl-CoA: malate CoA- 2.8.3.— Chloroflexusaurantiacus transferase succinate dehydrogenase 1.3.99.1 Chloroflexusaurantiacus (physiological acceptor unknown) fumarate hydratase 4.2.1.2Chloroflexus aurantiacus malyl-CoA lyase 4.1.3.24 Chloroflexusaurantiacus ribulose-1,5-bisphosphate 4.1.1.39 Chloroflexus aurantiacuscarboxylase CO dehydrogenase/acetyl-CoA 1.2.99.2 Chloroflexusaurantiacus synthase pyruvate synthase 1.2.7.1 Chloroflexus aurantiacus2-oxoglutarate synthase 1.2.7.3 Chloroflexus aurantiacus ATP citrate(pro-3s)-lyase 4.1.3.8 Chloroflexus aurantiacus phosphoribulokinase2.7.1.19 Chloroflexus aurantiacus malate-CoA ligase 6.2.1.9 Chloroflexusaurantiacus succinate-CoA ligase 6.2.1 3 Chloroflexus aurantiacusisocitrate lyase 4.1.3.1 Chloroflexus aurantiacus

Example 64 Engineered 3HP/4HB Cycle

A host cell of interest is engineered to have a 3HP/4HB cycle (Berg etal., Science 318:1782 (2007)). In more preferred embodiments, the CalvinCycle is removed and the 3HP/4HB Cycle is engineered into the host.Preferably, the host cell is selected or engineered to havephotosynthetic properties.

TABLE 25 Example Organism, Enzyme gene(s) acetyl-CoA carboxylaseMetallosphaera sedula malonyl-CoA reductase Metallosphaera sedula(NADPH) malonate semialdehyde Metallosphaera sedula reductase (NADPH)3-hydroxypropionyl-CoA Metallosphaera sedula synthetase (AMP-forming)3-hydroxypropionyl-CoA Metallosphaera sedula dehydratase acryloyl-CoAreductase Metallosphaera sedula (NADPH) propionyl-CoA carboxylaseMetallosphaera sedula methylmalonyl-CoA epimerase Metallosphaera sedulamethylmalonyl-CoA mutase Metallosphaera sedula succinyl-CoA reductaseMetallosphaera sedula (NADPH) succinate semialdehyde Metallosphaerasedula reductase (NADPH) 4-hydroxybutyryl-CoA Metallosphaera sedulasynthetase (AMP-forming) 4-hydroxybutyryl-CoA Metallosphaera seduladehydratase crotonyl-CoA hydratase Metallosphaera sedula3-hydroxybutyryl-CoA Metallosphaera sedula dehydrogenase (NAD+)acetoacetyl-CoA b-ketothiolase Metallosphaera sedula pyruvate synthaseMetallosphaera sedula

Example 65 Engineered Limonene Production

A host cell of interest is engineered to produce limonene. Preferably,the host cell is selected or engineered to have photosyntheticproperties. In more preferred embodiments, the Calvin Cycle is removedand the 3HP/4HB Cycle is engineered into the host.

TABLE 26 Enzyme EC # Product Mevalonate pathway to (R)- or (S)-limonene(4S)-limonene synthase 4.2.3.16 (S)-limonene (piney) (R)-limonenesynthase 4.2.3.20 (R)-limonene (citrus) geranyl-diphosphate synthase2.5.1.1 geranyl-PP isopentenyl-diphosphate 5.3.3.2 dimethylallyl-PDelta-isomerase diphosphomevalonate 4.1.1.33 isopentenyl-PPdecarboxylase phosphomevalonate kinase 2.7.4.2 mevalonate-5-PPmevalonate kinase 2.7.1.36 mevalonate-5-P 3-hydroxy-3-methylglutaryl-CoA1.1.1.34 mevalonate reductase Non-mevalonate pathway to (R)- or(S)-limonene (4S)-limonene synthase 4.2.3.16 (S)-limonene (piney)(R)-limonene synthase 4.2.3.20 (R)-limonene (citrus) geranyl-diphosphatesynthase 2.5.1.1 geranyl-PP isopentenyl-diphosphate 5.3.3.2dimethylallyl-P Delta-isomerase 4-hydroxy-3-methylbut-2-enyl 1.17.1.2isopentenyl-PP diphosphate reductase 4-hydroxy-3-methylbut-2-en-1-yl1.17.4.3 1-hydroxy-2-methyl- diphosphate synthase 2-butenyl-4-diphosphate 2-C-methyl-D-erythritol 4.6.1.12 2-C-methyl-D-2,4-cyclodiphosphate synthase erythritol-2,4- cyclodiphosphate4-(cytidine 5′-diphospho)-2-C- 2.7.1.148 2-phospho-4-(cytidinemethyl-D-erythritol kinase 5′-diphospho)-2-C- methyl-D-erythritol2-C-methyl-D-erythritol 2.7.7.60 4-(cytidine 5′- 4-phosphatediphospho)-2-C- cytidylyltransferase methyl-D-erythritol1-deoxy-D-xylulose-5-phosphate 1.1.1.267 2-C-methyl-D- reductoisomeraseerythritol-4-P 1-deoxy-D-xylulose-5-phosphate 2.2.1.7 1-deoxy-D-synthase xylulose-5-P

Example 66 Enhanced Secretion of Fatty Acid Esters

Cloning of fadL and construction of vectors for expressing fadL incyanobacteria: fadL (SEQ ID NO: 24) was generated by PCR fromEscherichia coli (Migula) strain genomic DNA (ATCC #700926) usingPhusion™ Hot Start High-Fidelity DNA Polymerase (Developed &Manufactured By Finnzymes Oy and distributed by New England Biolabs,Ipswitch, Mass.)) according to manufacturer's instructions. The forwardprimer (fadl_FP) used in this reaction contains a 5′ NdeI restrictionsite and the reverse primer (fadl_RP) contains a 5′ EcoRI restrictionsite. Amplified DNA was subsequently purified by gel electrophoresisfollowed by agarose purification using the Qiagen gel extraction kitprotocol (Qiagen). Purified DNA product and vector pJB41 (a vector basedon the DNA 2.0 pJ204 vector containing NdeI and EcoRI cloning sites)were both digested with NdeI (New England Biolabs; Ipswitch, Mass.) andEcoRI (New England Biolabs; Ipswitch, Mass.) followed by ligationovernight using T4 ligase (New England Biolabs; Ipswitch, Mass.). Theligated construct was transformed into NEB EPI400 CopyCutter competentE. coli (New England Biolabs: Ipswitch, Mass.). Colonies weresubsequently grown in LB media and screened for insert by PCR using thesame primers as above. The selected construct was validated by sequenceverification GENEWIZ (South Plainfield, N.J.).

This fadL insert was cloned into a recombination vector suitable forinsertion into Synechococcus sp. 7002 containing several elements. Anupstream homology region (UHR; see, e.g., SEQ ID NO: 26) and adownstream homology region (DHR; see, e.g., SEQ ID NO:26) is present inthe fadL insert allowing recombination into pAQ7 (pAQ7 plasmid sequence,see Genbank #CP000957). The homology regions flank a multiple cloningsite (mcs) and a kanamycin cassette which provides resistance in both E.coli and Synechococcus sp. 7002. An XbaI site present upstream of theUHR and downstream of the DHR allows linearization of the fadL insert tofacilitate transformation and integration into the host organism.

Several variants bearing fadL under different strength promoters havebeen prepared. One such promoter (amt1 from JCC160) is presented in SEQID NO: 26. Other variants that have been constructed include constructswith fadL under the control of Thermosynechococcus elongatus BP-1promoters such as tsr2142 or psaA.

Assessment of fadL expression constructs. The fadL constructs will beintegrated onto pAQ7 of the engineered Synechococcus strain JCC803(pAQ1: lacIq Ptrc-tesA-fadD-wax synthase-aadA) using the followingprotocol. The strain will be grown from colonies in an incubated shakerflask at 37° C. at 2% CO₂ to an OD₇₃₀ of 11n A⁺ medium supplemented with200 mg/L spectinomycin. 500 μL of culture will be added to amicrocentrifuge tube with 50 μL of 1-5 μg of DNA prepped from a QiagenQiaprep Spin Miniprep Kit (Valencia, Calif.) for each construct afterdigesting with xbaI (New England Biolabs; Ipswitch, Mass.). Cells willbe incubated in the dark at 37° C. for four hours in a shakingincubator. 200 μL of cells will be plated on A⁺ medium plates with 1.5%agarose and 100 μg/ml spectinomyin and incuated at 37° C. for 24 h underlight. 50 μg/mL of kanamycin will be underlayed on the plates, andresistant colonies will be visible in 7-10 days.

Expression and secretion of ethyl ester in strains containing the fadLexpression constructs will be measured and compared as described in,e.g., Example 17, herein. In addition, cultures will be partitioned withan organic solvent such as isooctane or ethyl acetate, and the organicsolvent partition will be analyzed by GC/MS as detailed in example 17 todetect and quantify esters present in the media. Other techniques knownto those skilled in the art may also be used to measure the amount ofethyl ester secreted into the medium.

Different levels of expression and/or secretion may be desired,depending on the particular environment and/or purpose for theexpression and/or secretion. A variety of constructs effecting a rangeof expression levels is therefore desirable. In addition to fadL from E.coli, other transporters including the CER5 wax transporter from A.thaliana (Q9C8K2; SEQ ID NO: 23) and XylN from Pseudomonas putida(Q8VMI2; SEQ ID NO:25) will be incorporated into constructs and theirenhancing effects on ethyl ester secretion will be measured. In certainembodiments, two or more distinct transporters may be incorporated intoa single strain to achieve levels of biofuel secretion that are greaterthan the levels achieved using a single transporter. In addition,recombinant transporters may be modified or mutated to modulate(increase or decrease) the level of secretion achieved, or to alter thespecificity of the transporter(s) such that the transport of additionaltypes of molecules is increased or decreased relative to the unmodifiedform of the transporter. Such modified transporters may have amino acidsequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to the transporters disclosed herein. Other promoters inaddition to those mentioned above may be used in this context, includingapcA, EM7, tRNA-Glu, lacIq-Ptrc, nblA, hsp33, rbcL, cpcC, cpcB, rpsU,PaphII, Pci and nirA. As with the transporter sequences, modificationsmay be made to any of the promoter sequences to modulate the level ofexpression of the transporters under their control.

Example 67 Enhanced Ethanol Production

This Example provides an illustration of how ethanol production in anengineered organism can be improved and made more efficient by modifyingthe expression levels and/or identity of enzymes involved in ethanolproduction. Specifically, a series of pAQ7 Δldh targeting plasmids wereconstructed containing Moorella alcohol dehydrogenase (adh) genes underthe control of different strength promoters. These plasmids weretransformed into JCC445 (see Table 5) and the resulting strains wereexamined for ethanol production. Transformation of JCC445 by severaldifferent plasmids was shown to enhance production of ethanol but onlyone plasmi, PcI_adhAM, resulted in increased levels of ethanolaccompanied by a concomitant decrease in acetaldehyde levels.

Strain Construction:

TABLE 27 Strain Host+ Plasmid (pAQ7-derived) JCC445 + 773 JCC445 + 773Δldh_kan_PaphII_adhAM JCC445 + 782 JCC445 + 782 Δldh_kan_PcI_adhAM (akaJCC1005) (SEQ ID NO: 27)

Four μg of the plasmid DNA was digested with XmaI and used to transform400 μl of JCC445 (OD₇₃₀=1.15) using the transformation protocoldescribed herein. The entire transformation mix was plated onto A⁺ agarplates and incubated at 37 C (˜50 μE) in air. After 24 hours the plateswere underplated with kanamycin to a final concentration of 50 μg/ml andincubated as above. After 5 days background growth was diminished andKm^(R) colonies began to be visible, at which point the plates weretransferred to 37 C (˜50 μE)+1% CO₂. After 3 days, two Km^(R) coloniesfrom each transformation were streaked onto A^(+Km50Sp100) agar platesfor single colony isolation. Streaked plates were incubated for 4 daysat 37 C (˜50 μE) in air and then transferred to 37 C (˜50 μE)+1% CO₂ foran additional 3 days.

Batch Ethanol in Flasks: A single colony of one clone from the originalstreak plate from each transformation was inoculated into 5 mlA^(+Km50Sp100) broth in a 16 mM×150 mM plastic-capped culture tube andincubated in the Infors incubator (37 C, 150 rpm, 2% CO₂) at a ˜60°angle. After 3 days the cultures were transferred into foam-plugged125-ml Erlenmeyer flasks containing 30 ml JB2.1^(Km50Sp100) to anOD₇₃₀=˜0.1. The initial weight of each flask culture was determined sothat sterile dH₂O could be added at sampling times to account forevaporation. At each sampling point, 300 μl of culture was removed andthe cultures were replenished with an equal amount of fresh JB2.1medium. At roughly 24 hour intervals, samples were taken for growth rateand ethanol/acetaldehyde determinations. OD₇₃₀ measurements and derivedgrowth curves are shown FIG. 22. The levels of ethanol and acetaldehydein the media were determined as described herein and the data ispresented in FIG. 23 and FIG. 24, respectively.

As can be seen from the Figures, the levels of ethanol were highest inJCC445+782, where additional adhA_(M) is expressed under the control ofthe lambda cI promoter. The cumulative levels of ethanol produced bythis strain were approximately 4 g/L. At any given time point, themeasured levels of ethanol were substantially higher than those measuredin JC445 or JCC445+773, in some cases nearly twice as high. In addition,the same strain produced only 50% (or less) acetaldehyde compared toJC445 or JCC445+773 (expressing adhA_(M) under the control of the PaphIIpromoter). Thus, this Example demonstrates not only that enhanced levelsof ethanol and reduced levels of toxic intermediates can result fromalterations to promoters controlling adhA_(M) expression, but alsoprovides a specific example of an engineered cyanobacteria (JCC445+773)with enhanced ethanol producing capabilities.

All publications, patent documents and sequences referred to herein arehereby incorporated by reference in their entirety for all purposes tothe same extent as if each were so individually denoted.

What is claimed is:
 1. A method for production of ethanol, comprising:culturing an engineered cyanobacterium in a culture medium in thepresence of light and inorganic carbon, wherein said engineeredcyanobacterium comprising a recombinant pyruvate decarboxylase nucleicacid sequence encoding a pyruvate decarboxylase having EC 4.1.1.1 and aplurality of recombinant alcohol dehydrogenase nucleic acid sequencesencoding an alcohol dehydrogenase having EC 1.1.1.1 or EC 1.1.1.2,wherein the expression level the alcohol dehydrogenase and theexpression level of the pyruvate decarboxylase are regulated by separatepromoters, wherein the expression level of alcohol dehydrogenase in theengineered cyanobacterium is greater than the expression level ofalcohol dehydrogenase in a control engineered cyanobacterium comprisinga recombinant alcohol dehydrogenase nucleic acid sequence and arecombinant pyruvate decarboxylase nucleic acid sequence regulatedtogether by a single promoter, and wherein the engineered cyanobacteriumproduces ethanol in an amount greater than a non-engineeredcyanobacterium, when cultured under identical conditions.
 2. The methodof claim 1, wherein different promoters are used to achieve differentialexpression of each recombinant nucleic acid sequence.
 3. The method ofclaim 2, wherein one of said recombinant nucleic acid sequences iscontrolled by an inducible promoter and one of said recombinant nucleicacid sequences is modulated by a constitutive promoter.
 4. The method ofclaim 3, wherein the expression of said recombinant alcoholdehydrogenase nucleic acid sequence is modulated by a constitutivepromoter.
 5. The method of claim 3, wherein the expression of saidrecombinant pyruvate decarboxylase nucleic acid sequence is modulated bya chemically-affected promoter.
 6. The method of claim 5, wherein theexpression of said recombinant pyruvate decarboxylase nucleic acidsequence is modulated by an ammonia-repressible promoter.
 7. The methodof claim 6, wherein said inducible promoter is nirA.
 8. The method ofclaim 1, wherein said recombinant alcohol dehydrogenase nucleic acidsequence and said recombinant pyruvate decarboxylase nucleic acidsequence are encoded by (1) the same plasmid, or (2) the samechromosome, or (3) separate plasmids, or (4) separately on a chromosomeand a plasmid.
 9. The method of claim 1, wherein the activity of alcoholdehydrogenase is varied by controlling the level of a co-factor requiredby alcohol dehydrogenase.
 10. The method of claim 1, wherein saidcyanobacterium is a thermophilic cyanobacterium.
 11. The method of claim1, wherein said recombinant alcohol dehydrogenase gene encodes aMoorella alcohol dehydrogenase.
 12. An engineered cyanobacteriumcomprising a recombinant pyruvate decarboxylase nucleic acid sequenceencoding a pyruvate decarboxylase having EC 4.1.1.1 and a plurality ofrecombinant alcohol dehydrogenase nucleic acid sequences encoding analcohol dehydrogenase having EC 1.1.1.1 or EC 1.1.1.2, wherein theexpression level the alcohol dehydrogenase and the expression level ofthe pyruvate decarboxylase are regulated by separate promoters, whereinthe expression level of alcohol dehydrogenase in the engineeredcyanobacterium is greater than the expression level of alcoholdehydrogenase in a control engineered cyanobacterium comprising arecombinant alcohol dehydrogenase nucleic acid sequence and arecombinant pyruvate decarboxylase nucleic acid sequence regulatedtogether by a single promoter, and wherein the engineered cyanobacteriumproduces ethanol in an amount greater than a non-engineeredcyanobacterium, when cultured under identical conditions.
 13. Theengineered cyanobacterium of claim 12, wherein the expression of alcoholdehydrogenase is driven by different promoters.
 14. The engineeredcyanobacterium of claim 12, wherein said cyanobacterium lacks afunctioning lactate dehydrogenase gene or lactate dehydrogenase enzymeactivity.
 15. The engineered cyanobacterium of claim 12, wherein saidcyanobacterium is a thermophile or a strain of Synechococcus.
 16. Theengineered cyanobacterium of claim 13, wherein one of said recombinantnucleic acid sequences are controlled by an inducible promoter and oneof said recombinant nucleic acid sequence is modulated by a constitutivepromoter.
 17. The engineered cyanobacterium of claim 16, wherein theexpression of said recombinant alcohol dehydrogenase nucleic acidsequence is modulated by a constitutive promoter.
 18. The engineeredcyanobacterium of claim 16, wherein the expression of said recombinantpyruvate decarboxylase nucleic acid sequence is modulated by achemically-affected promoter.
 19. The engineered cyanobacterium of claim18, wherein the expression of said recombinant pyruvate decarboxylasenucleic acid sequence is modulated by an ammonia-repressible promoter.20. The engineered cyanobacterium of claim 19, wherein said induciblepromoter is nirA.
 21. The engineered cyanobacterium of claim 12, whereinsaid recombinant alcohol dehydrogenase nucleic acid sequence and saidrecombinant pyruvate decarboxylase nucleic acid sequence are encoded by(1) the same plasmid, or (2) the same chromosome, or (3) separateplasmids, or (4) separately on a chromosome and a plasmid.
 22. Theengineered cyanobacterium of claim 12, wherein the activity of alcoholdehydrogenase is varied by controlling the level of a co-factor requiredby alcohol dehydrogenase.
 23. The engineered cyanobacterium of claim 15,wherein said cyanobacterium is a thermophilic cyanobacterium.
 24. Theengineered cyanobacterium of claim 12, wherein said recombinant alcoholdehydrogenase nucleic acid sequence encodes a Moorella alcoholdehydrogenase.